Inhibitors of glucosylceramide synthase

ABSTRACT

The hemitartrate salt of a compound represented by the following structural formula: 
     
       
         
         
             
             
         
       
     
     (Formula 1 Hemitartrate), which may be used in pharmaceutical applications, are disclosed. Particular single crystalline forms of the Formula (I) Hemitartrate are characterized by a variety of properties and physical measurements. As well, methods of producing crystalline Formula (I) Hemitartrate, and using it to inhibit glucosylceramide synthase or lowering glycosphingolipid concentrations in subjects to treat a number of diseases, are also discussed. Pharmaceutical compositions are also described.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/143,821, filed Jan. 7, 2021, which is a continuation of U.S. patentapplication Ser. No. 15/049,946, filed Feb. 22, 2016 (now U.S. Pat. No.10,888,547), which is a continuation of U.S. patent application Ser. No.14/994,489, filed Jan. 13, 2016 (now abandoned), which is a continuationof U.S. patent application Ser. No. 13/511,768, which adopts theInternational filing date of Nov. 24, 2010, which is a 35 U.S.C. § 371national stage filing of International Application No.PCT/US2010/057952, filed Nov. 24, 2010, which claims priority to U.S.Provisional Application No. 61/264,748, filed Nov. 27, 2009, thedisclosures of which are herein incorporated by reference in theirentirety.

BACKGROUND

Glycosphingolipids (GSLs) are a class of naturally-occurring compoundswhich have a multitude of biological functions, including the ability topromote cell growth, cell differentiation, adhesion between cells orbetween cells and matrix proteins, binding of microorganisms and virusesto cells, and metastasis of tumor cells. GSLs are derived fromglucosylceramide (GlcCer), which is produced from ceramide andUDP-glucose by the enzyme UDP-glucose: N-acylsphingosineglucosyltransferase (GlcCer synthase). The structure of ceramide isshown below:

The accumulation of GSLs has been linked to a number of diseases,including Tay-Sachs, Gaucher, and Fabry diseases (see, for example, U.S.Pat. No. 6,051,598). GSLs have also been linked to certain cancers. Forexample, it has been found that certain GSLs occur only in tumors or atabnormally high concentrations in tumors; exert marked stimulatory orinhibitory actions on tumor growth when added to tumor cells in culturemedia; and inhibit the body's normal immunodefense system when shed bytumors into the surrounding extracellular fluid. The composition of atumor's GSLs changes as the tumors become increasingly malignant andantibodies to certain GSLs inhibit the growth of tumors.

Compounds which inhibit GlcCer synthase can lower GSL concentrations andhave been reported to be useful for treating a subject with one of theaforementioned diseases. A number of potent inhibitors of GlcCer,referred to herein as “amino ceramide-like compounds”, are disclosed inU.S. Pat. Nos. 6,051,598, 5,952,370, 5,945,442, 5,916,911 and 6,030,995.The compound of Formula (I), shown below, is a GlcCer synthase inhibitorcurrently in clinical trials for the treatment of Gaucher disease:

There is a need for salt forms of this drug candidate that arecrystalline and otherwise have physical properties that are amenable tolarge scale manufacture. There is also a need for pharmaceuticalformulations in which this drug candidate is stable and effectivelydelivered to the patient, as well as improved treatment methodsutilizing this compound.

SUMMARY OF THE INVENTION

It has been found that the hemitartrate salt of the compound of Formula(I) (hereinafter “Formula (I) Hemitartrate”) can be crystallized underwell-defined conditions to provide certain non-hygroscopic crystallineforms. Formula (I) Hemitartrate has several advantageous properties whencompared to other salts of Formula (I). As described further in Example1, many Formula (T) salts, including citrate, malate, fumaric,methylsulfonic, and acetic, could not be obtained in solid form.Although the hydrochloric and 1:1 tartrate salt of Formula (I) wereobtained in solid form, neither were crystalline and both were toohydroscopic for formulation. Formula (I) Hemitartrate is easier toformulate and synthesize than the free base and the other salts. Formula(I) Hemitartrate is also crystalline, non-hydroscopic, water-soluble andflows better than the corresponding free base (hereinafter “Formula (I)Free Base”) and other salts. Thus, these favorable properties makeFormula (I) Hemitartrate amenable to large scale manufacture as a drugcandidate.

It has also been found that stable granules for capsule formulations ofFormula (I) Hemitartrate can be prepared using defined ratios of a waterinsoluble filler, a water soluble filler and Formula (I) Hemitartrate.Based on this discovery, stable pharmaceutical formulations of Formula(I) Hemitartrate are disclosed.

It has also been found that the compound of Formula (I) orpharmaceutically acceptable salts thereof (including Formula (I)Hemitartrate) are metabolized by the liver, primarily by cytochrome P450enzymes. Based on this discovery, methods of treatment with the compoundof Formula (I) or pharmaceutically acceptable salts thereof (includingFormula (I) Hemitartrate) that reduce the potential for drug/druginteractions are disclosed.

It has also been found that Gaucher mice administered recombinantglucocerebrosidase and then Formula (I) Hemitartrate showed lower levelsof GL1 in visceral organs and a reduced number of Gaucher cells in theliver compared with treatment with glucocerebrosidase alone or Formula(I) Hemitartrate alone. Based on this discovery, combination therapieswith the compound of Formula (I) or pharmaceutically acceptable saltsthereof (including Formula (I) Hemitartrate) are also disclosed.

One embodiment of the present application is the hemitartrate salt ofthe compound represented by Formula (I). As noted above, thehemitartrate salt of the compound represented by Formula (I) is referredto herein as “Formula (I) Hemitartrate.” The compound represented byFormula (I) is referred to herein as “Formula (I) Free Base.”

Another embodiment of the present application provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand Formula (I) Hemitartrate.

Another embodiment provides a method of inhibiting glucosylceramidesynthase or lowering glycosphingolipid concentrations in a subject inneed thereof by administering to the subject an effective amount ofFormula (I) Hemitartrate.

Another embodiment provides the use of Formula (I) Hemitartrate for themanufacture of a medicament for inhibiting glucosylceramide synthase orlowering glycosphingolipid concentrations in a subject in need thereof.

Another embodiment provides the use of Formula (I) Hemitartrate forinhibiting glucosylceramide synthase or lowering glycosphingolipidconcentrations in a subject in need thereof.

Another embodiment is a method of treating a subject with Gaucherdisease. The method comprises administering to the subject an effectiveamount of a first therapeutic agent in combination with an effectiveamount of a second therapeutic agent. The first therapeutic agent isrepresented by Formula (I) or a pharmaceutically acceptable saltthereof; and the second therapeutic agent is effective for the treatmentof Gaucher disease.

Another embodiment is a method of treating a subject with Fabry disease.The method comprises administering to the subject an effective amount ofa first therapeutic agent in combination with an effective amount of asecond therapeutic agent. The first therapeutic agent is represented byFormula (I) or a pharmaceutically acceptable salt thereof; and thesecond therapeutic agent is effective for the treatment of Fabrydisease.

Another embodiment provides pharmaceutical composition comprising:

the hemitartrate salt of a compound represented by Formula (I); at leastone water-soluble filler; at least one water-insoluble filler; at leastone binder; and at least one lubricant.

Another embodiment of the invention is a method of treating a subjectwith Fabry disease. The method comprises the steps of:

-   -   a) administering to the subject an effective amount of a        compound of Formula (I), or a pharmaceutically acceptable salt        thereof;    -   b) testing the subject to determine whether the subject is a        poor, intermediate or extensive/ultra rapid P450 metabolizer;    -   c) if the subject is an intermediate or extensive/ultra rapid        P450 metabolizer, determining an adjusted effective amount of        the compound; and    -   d) administering to the subject an adjusted effective amount of        the compound of Formula (I) if the subject is an intermediate or        extensive/ultra rapid P450 metabolizer and administering to the        subject an effective amount of the compound of Formula (I) if        the subject is a poor P450 metabolizer.

Another embodiment of the invention is a method of treating a subjectwith Gaucher disease. The method comprises the steps of:

-   -   a) administering to the subject an effective amount of a        compound of Formula (I), or a pharmaceutically acceptable salt        thereof;    -   b) testing the subject to determine whether the subject is a        poor, intermediate or extensive/ultra rapid P450 metabolizer;    -   c) if the subject is an intermediate or extensive/ultra rapid        P450 metabolizer, determining an adjusted effective amount of        the compound; and    -   d) administering to the subject an adjusted effective amount of        the compound of Formula (I) if the subject is an intermediate or        extensive/ultra rapid P450 metabolizer and administering to the        subject an effective amount of the compound of Formula (I) if        the subject is a poor P450 metabolizer.

Another embodiment of the invention is a method of treating a subjectwith Fabry disease. The method comprises the steps of:

-   -   a) administering to the subject an effective amount of a        compound represented by the following structural formula:

or a pharmaceutically acceptable salt thereof;

-   -   b) assessing trough plasma levels of the compound in the        subject; and    -   c) adjusting the amount of compound administered to the subject        so that the trough plasma levels of the compound are at least 5        ng/ml. Alternatively, the trough plasma levels and C_(max) of        the compound in the subject are assessed in step b) and in        step c) the amount of compound administered to the subject is        adjusted so that trough plasma levels of the compound in the        subject are at least 5 ng/ml and the C_(max) of the compound in        the subject is below 100 ng/ml.

Another embodiment of the invention is a method of treating a subjectwith Gaucher disease. The method comprises the steps of:

-   -   a) administering to the subject an effective amount of a        compound represented by the following structural formula:

or a pharmaceutically acceptable salt thereof;

-   -   b) assessing trough plasma levels of the compound in the        subject; and    -   c) adjusting the amount of compound administered to the subject        so that the trough plasma levels of the compound in the subject        are least 5 ng/ml. Alternatively, the trough plasma levels and        C_(max) of the compound in the subject are assessed in step b)        and in step c) the amount of compound administered to the        subject is adjusted so that trough plasma levels of the compound        in the subject are at least 5 ng/ml and the C_(max) of the        compound in the subject is below 100 ng/ml

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the experimental XRPD pattern (room temperature) forFormula (I) Hemitartrate.

FIG. 2 is a graph of the efficacy of enzyme and substrate reductiontherapies at lowering glucosylceramide levels in the liver of Gauchermice. Liver GL1 levels were measured in untreated 3 month-old Gauchermice (A) and following 2 weeks of treatment with recombinantglucocerebrosidase (B). Mice treated with recombinant glucocerebrosidasewere analyzed 10 weeks later without further treatment (C) or aftertherapy with Formula (I) Hemitartrate (D) at 150 mg/kg feed. GL1 levelsin the liver of mice administered Formula (I) Hemitartrate alone for theentire period of study (E) and in untreated, age-matched controls (F)are also shown. Data are expressed as means f standard error of the mean(SEM) (n=5). Statistical significance was determined using the unpairedt test.

FIG. 3 is a graph of the efficacy of enzyme and substrate reductiontherapies at lowering glucosylceramide levels in the spleen of Gauchermice. Spleen GL1 levels were measured in untreated 3 month-old Gauchermice (A) and following 2 weeks of treatment with recombinantglucocerebrosidase (B). Mice treated with recombinant glucocerebrosidasewere analyzed 10 weeks later without further treatment (C) or aftertherapy with Formula (I) Hemitartrate (D). GL1 levels in the spleen ofmice administered Formula (I) Hemitartrate alone for the entire periodof study (E) and in untreated, age-matched controls (F) are also shown.Data are expressed as means t standard error of the mean (SEM) (n=5).Statistical significance was determined using the unpaired t test.

FIG. 4 is a graph of the efficacy of enzyme and substrate reductiontherapies at lowering glucosylceramide levels in the lung of Gauchermice. Lung GL1 levels were measured in untreated 3 month-old Gauchermice (A) and following 2 weeks of treatment with recombinantglucocerebrosidase (B). Mice treated with recombinant glucocerebrosidasewere analyzed 10 weeks later without further treatment (C) or aftertherapy with Formula (I) Hemitartrate (D). GL1 levels in the lung ofmice administered Formula (I) Hemitartrate alone for the entire periodof study (E) and in untreated, age-matched controls (F) are also shown.Data are expressed as means±standard error of the mean (SEM) (n=5).Statistical significance was determined using the unpaired t test.

FIG. 5 is a graph showing the quantitation of the extent of CD68staining in the liver. The extent of CD68-positive staining on the liversections was quantified using MetaMorph software. Shown are levels inuntreated 3 month-old Gaucher liver (A) or following treatment withglucocerebrosidase (B). Mice treated with enzyme and then analyzed 10weeks later without further therapeutic intervention (C) or aftertherapy with Formula (I) Hemitartrate (D) are also illustrated. Theextent of staining in the liver of Gaucher mice administered Formula (I)Hemitartrate alone (E) and in untreated, age-matched control mice (F)are also shown. The data was collated from an analysis of ten 400×images per section from each of the mice. Statistical significance wasdetermined using the unpaired t test.

FIG. 6 is a graph that shows the efficacy of Formula (I) Hemitartrate inyoung D409V/null mice. Formula (I) Hemitartrate was administered to10-week-old D409V/null mice daily by oral gavage at a dose of 75 or 150mg/kg for 10 weeks. Glucosylceramidc levels in liver, lung, vasculatureand spleen were evaluated at the end of the study by HP-TLC. Data arepresented as a percentage of GL-1 in untreated age-matched control mice.Dashed lines indicate glucosylceramide levels observed in normal wildtype mice. *p<0.05; **p<0.01 relative to untreated control (two-tailed,unpaired t-test). Data are represented as means+standard error of themean (SEM) n=5 for 75 mg/kg; n=6 for 150 mg/kg).

FIG. 7 shows the effect of Formula (I) Hemitartrate therapy on theaccumulation of GL-3 in Fabry mouse liver, heart, kidney, spleen, brain,and blood.

FIG. 8 shows a graph of the effect of Formula (I) Hemitartrate therapyon the onset and progression of peripheral neuropathy in Fabry mice.

FIG. 9 shows graphs of measurements of some markers of kidney functionin Fabry mice treated with Formula (I) Hemitartrate.

FIG. 10 shows a timeline for ERT and SRT studies of mouse populationsreceiving different drug therapies: A) Fabrazyme bimonthly, no Formula(I) Hemitartrate; B) Fabrazyme bimonthly and Formula (I) Hemitartrate infood; C) Fabrazyme administered at the beginning of the study and atmonth four of the study and Formula (I) Hemitartrate in food; D) noFabrazyme, Formula (I) Hemitartrate in food; and E) no drug therapy.

FIG. 11 shows graphs of blood GL-3 levels in ng/mL of blood in sixpopulations (n=?) of mice (A-E Fabry-Rag; and F wild-type); the micepopulations received the following therapies: A) Fabrazyme bimonthly, noFormula (I) Hemitartrate; B) Fabrazyme bimonthly and Formula (I)Hemitartrate in food; C) Fabrazyme administered at the beginning of thestudy and at month four of the study and Formula (I) Hemitartrate infood; D) no Fabrazyme, Formula (I) Hemitartrate in food; E) no drugtherapy; and F) no drug therapy.

FIG. 12 shows graphs of GL-3 levels in Fabry-Rag mice liver and kidney;the mice populations (n=?) received the following therapies: A)Fabrazyme bimonthly, no Formula (I) Hemitartrate; B) Fabrazyme bimonthlyand Formula (I) Hemitartrate in food; C) Fabrazyme administered at thebeginning of the study and at month four of the study and Formula (I)Hemitartrate in food; D) no Fabrazyme, Formula (I) Hemitartrate in food;and E) no drug therapy

FIG. 13 shows graphs of urine GL-3 levels in Fabry-Rag mice; the micepopulations (n=?) received the following therapies: A) Fabrazymebimonthly, no Formula (I) Hemitartrate; B) Fabrazyme bimonthly andFormula (I) Hemitartrate in food; C) Fabrazyme administered at thebeginning of the study and at month four of the study and Formula (I)Hemitartrate in food; D) no Fabrazyme, Formula (I) Hemitartrate in food,and E) no drug therapy.

FIG. 14 is a graph showing the latency in seconds of heat sensitivity ofFabry-Rag mice receiving the following therapies: Fabrazyme bimonthly,no Formula (I) Hemitartrate; Fabrazyme bimonthly and Formula (I)Hemitartrate in food; Fabrazyme administered at the beginning of thestudy and at month four of the study and Formula (I) Hemitartrate infood; no Fabrazyme, Formula (I) Hemitartrate in food; no drug therapy;wild-trype mice; and untreated at three months.

FIG. 15 is a graph showing the total amount of degradation area of anHPLC trace of various blends comprising Formula (I) Hemitartrate,Lactose Monohydrate capsulating grade and Avicel PH 301(Microcrystalline cellulose) after having been exposed to 85° C. for 3days. The degradation area of the HPLC trace is ratio of the total areaof peaks corresponding to degradation relative to the total area ofpeaks corresponding to Formula (I) Hemitartrate and degradationproducts.

DETAILED DESCRIPTION OF THE INVENTION

The present application provides unique crystalline forms of Formula (I)Hemitartrate and new pharmaceutical compositions of Formula (I)Hemitartrate comprising the crystalline forms of Formula (I)Hemitartrate described herein. The present application also providesmethods of inhibiting glucosylceramide synthase or loweringglycosphingolipid concentrations in a subject in need thereof.Additionally, the present application provides methods for preparingspecific crystalline forms of Formula (I) Hemitartrate. The presentapplication also provides stable pharmaceutical formulations of Formula(I) Hemitartrate, combination therapies with the compound of Formula (I)or pharmaceutically acceptable salts thereof (including Formula (I)Hemitartrate) and methods of treatment with the compound of Formula (I)or pharmaceutically acceptable salts thereof (including Formula (I)Hemitartrate) that minimize the risk of drug/drug interactions.

Crystalline Forms of Formula (I) Hemitartrate

In a particular embodiment, at least a particular percentage by weightof Formula (I) Hemitartrate is crystalline. Particular weightpercentages include 70%, 72%, 75%, 77%, 80%, 82%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, ora percentage between 70% and 100%.

In another particular embodiment, at least a particular percentage byweight of Formula (I) Hemitartrate is a single crystalline form ofFormula (I) Hemitartrate. Particular weight percentages include 70%,72%, 75%, 77%, 80%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or a percentage between 70%and 100%.

As used herein, “crystalline” refers to a solid having a crystalstructure wherein the individual molecules have a highly homogeneousregular locked-in chemical configuration. Crystalline Formula (I)Hemitartrate can be crystals of a single crystalline form of Formula (I)Hemitartrate, or a mixture of crystals of different single crystallineforms. A single crystalline form means Formula (I) Hemitartrate as asingle crystal or a plurality of crystals in which each crystal has thesame crystal form.

When a particular percentage by weight of Formula (I) Hemitartrate is asingle crystalline form, the remainder of Formula (I) Hemitartrate issome combination of amorphous Formula (I) Hemitartrate, and/or one ormore other crystalline forms of Formula (I) Hemitartrate excluding thesingle crystalline form. When the crystalline Formula (I) Hemitartrateis defined as a specified percentage of one particular crystalline formof Formula (I) Hemitartrate, the remainder is made up of amorphous formand/or crystalline forms other than the one or more particular formsthat are specified. Examples of a single crystalline form include Form Aof Formula (I) Hemitartrate characterized by one or more properties asdiscussed herein.

Because tartaric acid has two carboxylic acid groups, it can form saltswith differing molar ratios of the compound represented by Formula (I)to tartrate (the conjugate base of tartaric acid). For example, the saltin which there is about a one to one molar ratio of tartrate to Formula(I) is Formula (I) Tartrate (1 tartrate: 1 Formula (I)); and the salt inwhich there is about a one to two molar ratio of tartrate to Formula (I)is Formula (I) Hemitartrate (1 tartrate: 2 Formula (I)).

The hemitartrate salt can exist in various stereoisomeric forms.Stereoisomers are compounds that differ only in their spatialarrangement. Enantiomers are pairs of stereoisomers whose mirror imagesare not superposable, most commonly because they contain anasymmetrically substituted carbon atom that acts as a chiral center.Diastereomers are stereoisomers that are not related as mirror images,most commonly because they contain two or more asymmetricallysubstituted carbon atoms.

When the stereochemistry is named (as in, for example, L-(+)-tartaricacid) or depicted by structure (as in, for example Formula (I)), thenamed or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99% or99.9% by weight pure relative to the other stereoisomers. When a singleenantiomer is named (as in, for example, L-(+)-tartaric acid) ordepicted by structure (as in, for example Formula (I)), the depicted ornamed enantiomer is at least 80%, 90%, 99% or 99.9% by weight opticallypure. Percent optical purity by weight is the ratio of the weight of theenantiomer over the weight of the enantiomer plus the weight of itsoptical isomer.

“Racemate” or “racemic mixture” means a compound of equimolar quantitiesof two enantiomers, wherein such mixtures exhibit no optical activity;i.e., they do not rotate the plane of polarized light.

Tartaric acid has three stereoisomers: L-(+)-tartaric acid ordextrotartaric acid and its enantiomer, levotartaric acid orD-(−)-tartaric acid, and the achiral form, mesotartaric acid. The L or Ddesignation does not indicate the acid's ability to rotate the plane ofpolarized light.

Any of the stereoisomers of tartaric acid can be used to prepare Formula(I) Hemitartrate. For example, the hemitartrate can be formed from onlyone of its stereoisomers, or a combination of them thereof. Thehemitartrate salt is selected from D-hemitartrate, L-hemitartrate,hemimesotartaric acid or racemic D,L-hemitartrate. In a specificembodiment, the hemitartrate salt is L-hemitartrate. “L-hemitartrate”means that the hemitartrate salt is formed from L-tartaric acid. RacemicD,L-hemitartrate means that both D-tartrate and L-tartrate were used inthe preparation of Formula (I) Hemitartrate. The amount of D-tartrate inracemic D,L-hemitartrate may be greater than, equal to, or less than theamount of L-tartrate present.

“Levorotatory” signifies that polarized light is rotated to the leftwhen passed through an asymmetric compound. The prefix to designatelevorotary is “L”.

“Dextrorotatory” signifies that polarized light is rotated to the rightwhen passed through an asymmetric compound. The prefix to designatelevorotary is “D”.

Preparation of Formula (I) Hemitartrate

Formula (I) Hemitartrate can be prepared by mixing Formula (I) withL-tartaric acid in a suitable solvent. Precipitation of Formula (I)Hemitartrate can be assisted by the addition of a seed crystal. Thesolvents that may be used are methanol, water, ethanol, acetone, ethylacetate, or combinations thereof.

The particular solid forms of Formula (I) Hemitartrate can be prepared,for example, by slow evaporation, slow cooling, and antisolventprecipitation. The solvents that may be used in these methods includewater, heptane, hexane, toluene, dichloromethane, ethanol, isopropylalcohol, acetonitrile, ethyl acetate, methanol, acetone, methyltertiary-butyl ether (referred to as “TBME” herein), p-dioxane, andtetrahydrofuran (referred to as “THF” herein).

Formula (I) Hemitartrate solid forms can be prepared by solventevaporation from a solution of Formula (I) Hemitartrate in a solvent ora solvent mixture. Suitable solvent mixtures include methanol, ethanol,acetone, water, ethyl acetate and dichloromethane. Preferred solventmixtures include ethanol, methanol, water and acetone.

Formula (I) Hemitartrate solid forms can be prepared through slowcooling of a heated solution of Formula (I) Hemitartrate in a solvent.Suitable solvents include ethanol, methanol, water, acetone, and ethylacetate.

Formula (I) Hemitartrate solid forms can be prepared through rapidcooling of a heated solution of Formula (I) Hemitartrate in a solvent,by placing the solution in an cooling bath. Suitable solvents includeethanol, methanol, acetone, water, ethyl acetate or mixtures of thesesolvents.

Formula (I) Hemitartrate solid forms can be prepared by adding asolution of Formula (I) Hemitartrate in a solvent as described above toan anti-solvent at a given temperature. More particularly, theanti-solvent is ethyl acetate, acetone, acetonitrile, toluene, THF,TBME, p-dioxane, isopropanol, or heptane. Particular solvent/antisolventmixtures include methanol/ethyl acetate, methanol/acetone,methanol/hexane, methanol/heptane, methanol/acetonitrile,methanol/toluene, methanol/THF, methanol/TBME, methanol/p-dioxane,ethanol/ethyl acetate, ethanol/hexane, ethanol/heptane, ethanol,acetone, ethanol/acetonitrile, ethanol/toluene, ethanol/TBME,ethanol/THF, water/THF, water/isopropanol, water/acetonitrile,water/acetone, dichloromethane/heptane, dichloromethane/acetone,dichloromethane/ethyl acetate, dichloromethane/acetonitrile,dichloromethane/toluene, dichloromethane/THF, dichloromethane/TBME,dichloromethane/p-dioxane, and dichloromethane/isopropanol.

Preferred solvent/antisolvent mixtures include methanol/ethyl acetate,methanol/acetone, methanol/TBME, and water/acetone.

As used herein, “anti-solvent” refers to a solvent, in which Formula (I)Hemitartrate has low solubility and cause the Hemitartrate toprecipitate out of solution in the form of fine powder or crystals.

Additional methods to generate the solid forms of Formula (I)Hemitartrate include precipitating the solid from ethyl acetate/acetoneand optionally drying solid formed at room temperature. In anothermethod, the solid can then be recrystallized from acetone with orwithout the addition of a seed crystal. Alternatively, Formula (I)Hemitartrate can be precipitated from ethyl acetate/acetone solvents andrecrystallized from ethyl acetate. Alternatively, Formula (I)Hemitartrate can then be recrystallized from isopropanol. AlternativelyFormula (T) Hemitartrate can be prepared using acetone only with nofurther recrystallization. Alternatively Formula (I) Hemitartrate can beprecipitated from acetone following a brief reflux, without furtherrecrystallization.

Alternatively, Formula (I) Hemitartrate can then be recrystallized frommethanol/acetone with or without the addition of a seed crystal.Alternatively, Formula (I) Hemitartrate can then be recrystallized fromwater/acetone with or without the addition of a seed crystal.

Characterization of Crystalline Forms of Formula (I) Hemitartrate

In a particular embodiment, the crystalline form of Formula (I)Hemitartrate, crystal Form A, is characterized by one, two, three, fouror five major XRPD peaks at 2θ angles of 5.1°, 6.6°, 10.7°, 11.0°,15.9°, and 21.7°. In an even more particular embodiment, the crystallineform is characterized by XRPD peaks at 2θ angles of 5.1°, 6.6°, 10.7°,11.0°, 13.30, 15.1°, 15.9°, 16.5°, 17.6°, 18.6°, 18.7°, 19.0°, 20.2°,21.7° and 23.5°. It is to be understood that a specified 20 angle meansthe specified value±0.2°.

As used herein, “major XRPD peak” refers to an XRPD peak with a relativeintensity greater than 25%. Relative intensity is calculated as a ratioof the peak intensity of the peak of interest versus the peak intensityof the largest peak.

Methods of Treatment Using Formula (I) Hemitartrate

As used herein, a subject is a mammal, preferably a human patient, butcan also be an animal in need of veterinary treatment, such as acompanion animal (e.g., dogs, cats, and the like), a farm animal (e.g.,cows, sheep, pigs, horses, and the like) or a laboratory animal (e.g.,rats, mice, guinea pigs, and the like). Subject and patient are usedinterchangeably.

One embodiment of the present application is a method of slowing, e.g.,inhibiting or reducing the activity of glucosylceramide synthase orlowering glycosphingolipid concentrations in a subject in need thereofby administering to the subject an effective amount of Formula (I)Hemitartrate salt, including crystalline forms thereof, as describedabove.

A subject in need of treatment is a subject with a condition or diseasethat benefits from inhibiting glucosylceramide synthase or loweringglycosphingolipid concentrations in the cells, particularly the lysosomeor the membrane of cells. Inhibitors of glucosylceramide synthase havebeen shown to be useful for treating lysosomal storage diseases such asTay-Sachs, Gaucher or Fabry disease (see, for example, U.S. Pat. Nos.6,569,889; 6,255,336; 5,916,911; 5,302,609; 6,660,749; 6,610,703;5,472,969; 5,525,616, the entire teachings of which are incorporatedherein by reference).

Examples of conditions or diseases include polycystic kidney disease andmembranous glomerulopathy (see U.S. Provisional Patent Applications61/130,401 and 61/102,541, the entire teachings of which areincorporated herein by reference), Glomerulonephritis andGlomerulosclerosis (See U.S. Provisional Patent Application 61/137,214)lupus (See PCT/US2009/001773, the entire teachings of which areincorporated herein by reference) diabetes, including type 2 diabetes(see WO 2006/053043, the entire teachings of which are incorporatedherein by reference); treating disorders involving cell growth anddivision, including cancer, collagen vascular diseases, atherosclerosis,and the renal hypertrophy of diabetic patients (see U.S. Pat. Nos.6,916,802 and 5,849,326, the entire teachings of which are incorporatedherein by reference); inhibiting the growth of arterial epithelial cells(see U.S. Pat. Nos. 6,916,802 and 5,849,326); treating patientssuffering from infections (see Svensson, M. et al., “EpithelialGlucosphingolipid Expression as a Determinant of Bacterial Adherence andCytokine Production,” Infect. and Immun., 62:4404-4410 (1994), theentire teachings of which are incorporated herein by reference);preventing the host, i.e., patient, from generating antibodies againstthe tumor (see Inokuchi, J. et al., “Antitumor Activity in Mice of anInhibitor of Glycosphingolipid Biosynthesis,” Cancer Lett.,38:23-30(1987), the entire teachings of which are incorporated herein byreference); and treating tumors (see Hakomori, S. “New Directions inCancer Therapy Based on Aberrant Expression of Glycosphingolipids:Anti-adhesion and Ortho-Signaling Therapy,” Cancer Cells 3:461-470(1991), Inokuchi, J. et al., “Inhibition of Experimental Metastasis ofMurine Lewis Long Carcinoma by an Inhibitor of Glucosylceramide Synthaseand its Possible Mechanism of Action,” Cancer Res., 50:6731-6737 (1990)and Ziche, M. et al., “Angiogenesis Can Be Stimulated or Repressed in InVivo by a Change in GM3:GD3 Ganglioside Ratio,” Lab. Invest., 67:711-715(1992), the entire teachings of which are incorporated herein byreference).

Formula (I) Hemitartrate can also be used for a cancer vaccine-likepreparation (see, for example, U.S. Pat. Nos. 6,569,889; 6,255,336;5,916,911; 5,302,609; 6,660,749; 6,610,703; 5,472,969; 5,525,616).

The compound of Formula (I) or a pharmaceutically acceptable saltthereof (including the hemitartrate salt thereof) can be used in thedisclosed methods as a mono-therapy, i.e., as the only pharmaceuticallyactive ingredient being administered to treat the indication.

Alternatively, the compound of Formula (I) or a pharmaceuticallyacceptable salt thereof (including the hemitartrate salt thereof) can beused in the disclosed methods as a combination therapy with othertherapeutically active drugs known in the art for treating the desireddiseases or indications. “Co-therapy” or “combination” or “combinationtherapy” or “co-administered” are used interchangeably herein and meanthat the compound of Formula (I) or pharmaceutically acceptable saltthereof (including the hemitartrate salt) is administered before, after,or concurrently with one or more other therapeutic agents. In oneembodiment, a combination therapy is used to treat a lysosomal diseasesuch as Gaucher disease or Fabry disease. Alternatively, the compound ofFormula (I) or pharmaceutically acceptable salt thereof (including thehemitartrate salt) is co-administered simultaneously (e.g.,concurrently) as either separate formulations or as a joint formulation.Alternatively, the agents can be administered sequentially, as separatecompositions, within an appropriate time frame, as determined by theskilled clinician (e.g., a time sufficient to allow an overlap of thepharmaceutical effects of the therapies). The compound of Formula (I) orpharmaceutically acceptable salt thereof (including the hemitartratesalt) and one or more other therapeutic agents can be administered in asingle dose or in multiple doses, in an order and on a schedule suitableto achieve a desired therapeutic effect.

Therapeutic agents effective for the treatment of Gaucher diseaseinclude glucocerebrosidase, analogues of glucocerebrosidase, inhibitorsof glucosylceramide synthase and molecular chaperones which bind toglucocerebrosidase and restore its correct conformation.Glucocerebrosidase or analogues thereof can be human or mammal derived.Alternatively, glucocerebrosidase and analogues thereof can be obtainedrecombinately. Analogues of glucocerebrosidase include truncated formsof the enzyme and/or enzymes with amino acid substitutions relative tothe native amino sequence of the native enzyme, provided that thebiological activity is retained. Examples of analogues ofglucocerebrosidase include Imiglucerase (sold under the tradenameCerezyme®) by Genzyme Corporation), Taliglucerase Alfa (to be marketedunder the tradename Uplyso® and developed by Protalix Biotherapeutics,Inc.) and Velaglucerase Alfa (developed by Shire PLC), which arerecombinant DNA-produced analogue of human β-glucocerebrosidase.Examples of molecular chaperones include isofagomine (in developmentunder the tradename Plicera™ by Amicus Therapeutics, Cranbury, N.J.).Isofagomine is also known as afegostat tartrate and contains thetartrate salt form of isofagomine as its active ingredient. Examples ofglucocerebrosidase inhibitors include miglustat (developed under thetradename of Zavesca™ by Actelion Pharmaceuticals Ltd. Allschwil,Switzerland).

Therapeutic agents effective for the treatment of Fabry disease includeα galactosidase A, analogues of α galactosidase A and molecularchaperones which bind to α galactosidase A and restore its correctconformation. α Galactosidase A or analogues thereof can be human ormammal derived. Alternatively, α galactosidase A and analogues thereofcan be obtained recombinantely. Analogues of α galactosidase A includetruncated forms of the enzyme and/or enzymes with amino acidsubstitutions relative to the native amino sequence of the nativeenzyme, provided that the biological activity is retained. Examples ofanalogues of α galactosidase A include Agalsidase beta (a recombinanthuman α-galactosidase sold under the tradename Fabrazyme® as afreeze-dried medicine by Genzyme Corporation) and Agalsidase alfa (arecombinant protein sold under the tradename Replagal® by Shire PLC).Examples of molecular chaperones include migalastat (developed under thetradename Amigal™ by Amicus Therapeutics, Cranbury, N.J. as a drugcontaining migalastat hydrochloride as its active ingredient).

In one embodiment, the combination therapy for the treatment of Gaucheror Fabry disease is carried out in two stages. In a first stage, a drugeffective for the treatment of Gaucher disease or Fabry disease(typically, glucocerebrosidase of an analogue thereof for Gaucherdisease and galactosidase A or an analogue thereof for Fabry disease) isused to stabilize the subject. For example, in Gaucher disease (or Fabrydisease), one of these drugs is used to reduce the burden of GL-1storage in the visceral organs such as in the liver, spleen, lung and/orkidney. Once this has been accomplished, the compound of Formula (I) ora pharmaceutically acceptable salt thereof (including the hemitartratesalt) is used in the second stage as a convenient maintenance therapy.The first stage typically lasts up to one, two, three or four weeks orup to one, two, three, four, six, nine or twelve months, or until thesubject's platelet count is equal to or greater than 100,000 mm³;hemoglobin concentration is equal to or greater than 11 g/dl (female) or12 g/dl (male); and/or the subject's spleen volume is less than or equalto 10 multiples of normal and liver volumes are less than or equal to1.5 multiples of normal. Administration of the first stage is typicallyended once therapy with the compound of Formula (I) is initiated.

As used herein, an “effective amount” refers to an amount effective toalleviate the existing symptoms of the subject being treated withminimal unacceptable side effects in the subject. The exact formulation,route of administration, and dosage is chosen by the individualphysician in view of the patient's condition. Dosage amount and intervalcan be adjusted individually to provide plasma levels of the activecompound that are sufficient to maintain desired therapeutic effects. Inaddition to the patient's condition and the mode of administration, thedose administered would depend on the severity of the patient's symptomsand the patient's age and weight. An effective amount will typicallyresult in plasma trough levels of the compound above at least 5 ng/ml.If plasma trough levels are below 5 ng/ml following administration of aneffective amount of the compound, the dose being administered to thatsubject is changed to an “adjusted effective amount” such that thetrough levels of the compound are at least 5 ng/ml. Alternatively, iftrough plasma levels of the compound are below 5 ng/ml and/or theC_(max) is above 100 ng/ml following administration of an effectiveamount of the compound, the dose being administered to the subject ischanged to an “adjusted effective amount” such that the trough plasmalevels of the compound are at least 5 ng/ml and the Ca is below 100ng/ml. Effective amounts can range from 0.1 to 500 mg/per day.Alternatively, the effective amount ranges from 50-300 mg/day. Inanother alternative, the effective amount ranges from 100-300 mg/day.The compound of the present application may be administered continuouslyor at specific timed intervals. For example, the compound of the presentapplication may be administered 1, 2, 3, or 4 times per day, such as,e.g., a daily or twice-daily formulation. Commercially available assaysmay be employed to determine optimal dose ranges and/or schedules foradministration.

In one embodiment, an effective amount for the compound of Formula (I)or a pharmaceutically acceptable salt thereof (including thehemitartrate salt described above) is (whether as a monotherapy or as aco-therapy) a daily dose of from 25 milligrams to 300 milligrams(alternatively 25 milligrams to 150 milligrams; in another alternativefrom 50 milligrams to 300 milligrams; and in another alternative from100 milligrams to 300 milligrams), such as 25, 50, 100, 200 or 300milligrams per day. In a specific embodiment, an effective amount of thecompound of Formula (I) or a pharmaceutically acceptable salt thereof(including Formula (I) Hemitartrate) is (whether as a monotherapy or asa co-therapy) a twice daily dose of 50 milligrams (for a total of 100milligrams per day), 100 milligrams (for a total of 200 milligrams perday) or 150 milligrams (for a total of 300 milligrams per day). In analternative embodiment, an effective amount for the compound of Formula(I) or a pharmaceutically acceptable salt thereof (including Formula (I)Hemitartrate) is (whether as a monotherapy or as a co-therapy)administered as a once daily dose of 100 milligrams/day, 200milligrams/day or 300 milligrams/day.

In another embodiment, an effective amount is determined is by assumingthat the subject is a “poor P450 metabolizer” and then assessing troughplasma levels and/or C_(max). The amount administered to the subject isthen changed to an adjusted effective amount, as described below, if thetrough plasma levels are below 5 ng/ml; or the trough levels of thecompound are below 5 ng/ml and/or the C_(max) is above 100 ng/ml; or ifthe subject is determined to be an intermediate or extensive/ultrarapidP450 metabolizer. An effective amount for poor P450 metabolizers is(whether as a monotherapy or as a co-therapy) commonly between 100-200milligrams per day, for example 100 or 200 milligrams, as a once dailydose or twice daily dose.

Typically, the pharmaceutical compositions of the present applicationcan be administered before or after a meal, or with a meal. As usedherein, “before” or “after” a meal is typically within two hours,preferably within one hour, more preferably within thirty minutes, mostpreferably within ten minutes of commencing or finishing a meal,respectively.

It has now been found that the compound of Formula (I) andpharmaceutically acceptable salts thereof (including Formula (I)Hemitartrate) is metabolized by the liver, primarily by cytochrome P450enzymes. Cytochrome P450s (“CYPs”) are the principal hepatic xenobioticmetabolizing enzymes. There are eleven xenobiotic-metabolizingcytochrome P450s expressed in a typical human liver (i.e., CYP1A2,CYP2A6, CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1 and CYP3A4/5). It has nowalso been found that CYP2D6 and CYP3A4 are the primary cytochrome P450isoforms that are responsible for de-toxifying the compound of Formula(I) and its pharmaceutically active salts, such as Formula (I)Hemitartrate. The level of activity of P450 enzymes differs according tothe individual. For example, individuals can be classified as poor,intermediate and extensive/ultra rapid P450 metabolizers. Because lowerlevels of P450 activity in an individual can give rise to drug/druginteractions (“DDI”), another embodiment of the invention is todetermine whether the subject is a poor, intermediate andextensive/ultra rapid P450 metabolizer. If the subject is anintermediate or extensive/ultra rapid metabolizer, then the doseadministered to that subject should be raised to an “adjusted effectivedose”, i.e., the amount which results in trough plasma levels of thecompound of at least 5 ng/ml; or the amount which results in troughlevels of the compound or at least 5 ng/ml and a C_(max) of the compoundbelow 100 ng/ml. The dose can raised incrementally and the subjectretested once, twice, three, four or as many times as necessary toachieve an adjusted effective dose.

For the CYP 2D6 gene there are four predicted phenotypes:

A “poor P450 metabolizer” carries two mutant alleles, which result incomplete loss of enzyme activity.

A, “intermediate P450 metabolizer” possess one reduced activity alleleand one null allele.

A “extensive P450 metabolizer” posses at least one and no more than twonormal functional alleles.

A “ultra rapid P450 metabolizer” carries multiple copies (3-13) offunctional alleles and produce excess enzymatic activity.

A subject is typically assessed as being a poor, intermediate orextensive/ultra rapid P450 metabolizer either through genotyping orthrough the monitoring of the trough plasma levels of a drug that ismetabolized by a P450 enzyme such as CYP2D6 or CYP3A4. Commonly, thetrough plasma levels and/or C_(max) of the compound of Formula (I) or apharmaceutically acceptable salt thereof, including Formula (I)hemitartrate are monitored in the subject for up to one, two, three orfour weeks, or up to one, two, three, six, nine or twelve months or morefollowing initiation of treatment with the compound. Adjustments to thedose are made, as necessary, to maintain the levels within the describedlimits, i.e., a trough plasma level at or above 5 ng/ml.

Subjects can become poor P450 metabolizers as a result of being treatedwith certain drugs that are P450 enzyme inhibitors. Examples of suchdrugs include paroxetine, fluoxetine, quinidine, or ketoconazole.Alternatively, a subject is a poor P450 metabolizer as a result of lowexpression of a P450 enzyme. In such instances, the low expression canbe assessed by determining P450 enzyme expression in the subject, i.e.,genotyping the subject for the P450 enzyme. For example, expression ofCYP2D6 is commonly assessed by PCR (McElroy et.al. “CYP2D6 Genotyping asan Alternative to Phenotyping for Determination of Metabolic Status in aClinical Trial Setting”, AAPS Pharmsi (2000) 2(4):article 33(http://www.pharmsci.org/)) or by microarray based pharmacogenomictesting (Background Information, Roche Diagnostics “The CYP450 GeneFamily and Drug Metabolism”, Hoffmann La Roche Ltd.), the entireteachings of which are incorporated herein by reference. As such, thesubject can be conveniently genotyped for P450 expression (e.g., CYP2D6)prior to the initiation of treatment and administered an adjustedeffective amount, if necessary. In the event of genotyping prior to theinitiation of treatment, it is still advisable to monitor trough plasmalevels and C_(max) of the compound and adjust the dose, as necessary.

Effective amounts for migalastat, agalsidase β, imiglucerase,isofagomine and miglustat are as described on the drug label or ascarried out in the clinical trials of each drug.

The compound of Formula (I) can react with pharmaceutically acceptableacids to form a pharmaceutically acceptable salt. Examples ofpharmaceutically acceptable acids included inorganic acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Pharmaceutical Compositions Including Formula (i) Hemitartrate

Suitable formulations and modes of administration for the compound ofFormula (I) or a pharmaceutically acceptable salt thereof (including thehemitartrate salt thereof) include those described in U.S. Pat. No.7,253,185, the entire teachings of which are incorporated herein byreference. A preferred formulation for Formula (I) Hemitartrate isdescribed in the following paragraphs.

One embodiment of the invention is a pharmaceutical compositioncomprising Formula (I) Hemitartrate, at least one water-soluble filler,at least one water-insoluble filler, at least one binder, and at leastone lubricant. Suitable water-soluble fillers can include, for example,anhydrous lactose, lactose monohydrate, mannitol, sodium chloride,powdered sugar, sorbitol, sucrose, inositol and pregelatinized starch.Suitable water-insoluble fillers can include, for example,microcrystalline cellulose, calcium phosphate and starch. Suitablebinders can include, for example, pre-gelatinized starch, sodiumcarboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, copolyvidone, gelatin, natural gums,starch paste, sucrose, corn syrup, polyethylene glycols and sodiumalginate. Suitable lubricants can include, for example, hydrogenatedvegetable oil, calcium stearate, and glyceryl behenate. In oneembodiment of the pharmaceutical composition, the water-soluble filleris selected from the group consisting of anhydrous lactose, lactosemonohydrate, mannitol, sodium chloride, powdered sugar, sorbitol,sucrose, inositol and pregelatinized starch; the water-insoluble filleris selected from the group consisting of microcrystalline cellulose,calcium phosphate and starch the binder is selected from the groupconsisting of pre-gelatinized starch, sodium carboxymethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, copolyvidone, gelatin, natural gums, starch paste, sucrose,corn syrup, polyethylene glycols and sodium alginate; and the lubricantis selected from the group consisting of hydrogenated vegetable oil,calcium stearate, and glyceryl behenate.

The pharmaceutical formula comprises between 8 wt % to 32 wt %, between8 wt % to 24 wt %, between 12 wt % to 20 wt % or between 14 wt % to 18wt/o of the water insoluble filler on a dry solids basis.

The pharmaceutical formula comprises between 26 wt % to 50 wt %, between30 wt % to 46 wt %, between 34 wt % to 46 wt % or between 38 wt % to 44wt % of the water soluble filler on a dry solids basis.

The pharmaceutical composition comprises between 30 wt % and 45 wt %,between 35 wt % and 40 wt % and 36 wt % to 39 wt % Formula (I)Hemitartrate on a dry solids basis.

The pharmaceutical formulation typically comprises between 2 wt % and 6wt % binder on a dry solids basis.

The pharmaceutical formulation typically comprises between 0.1 wt % and2 wt % binder on a dry solids basis.

In a specific embodiment, the pharmaceutical formula comprises between 8wt % to 32 wt % water insoluble filler, between 26 wt % to 50 wt %,water soluble filler, between 30 wt % and 45 wt % Formula (I)Hemitartrate, between 2 wt % and 6 wt % binder and between 0.1 wt % and2 wt % binder, all on a dry solids basis. More specifically, thewater-soluble filler is lactose monohydrate; and the water-insolublefiller is microcrystalline cellulose. Even more specifically thewater-soluble filler is lactose monohydrate; the water-insoluble filleris microcrystalline cellulose; the binder is hydroxypropylmethylcellulose; and the lubricant is glyceryl behenate.

In a specific embodiment, the pharmaceutical formula comprises between 8wt % to 32 wt % water insoluble filler, between 26 wt % to 50 wt %,water soluble filler, between 35 wt % and 40 wt % Formula (I)Hemitartrate, between 2 wt % and 6 wt % binder and between 0.1 wt % and2 wt/o binder, all on a dry solids basis. More specifically, thewater-soluble filler is lactose monohydrate; and the water-insolublefiller is microcrystalline cellulose. Even more specifically thewater-soluble filler is lactose monohydrate; the water-insoluble filleris microcrystalline cellulose; the binder is hydroxypropylmethylcellulose; and the lubricant is glyceryl behenate.

In another specific embodiment, the pharmaceutical formula comprisesbetween 8 wt % to 24 wt % water insoluble filler, between 30 wt % to 46wt %, water soluble filler, between 35 wt % and 40 wt % Formula (I)Hemitartrate, between 2 wt % and 6 wt % binder and between 0.1 wt % and2 wt % binder, all on a dry solids basis. More specifically, thewater-soluble filler is lactose monohydrate; and the water-insolublefiller is microcrystalline cellulose. Even more specifically thewater-soluble filler is lactose monohydrate; the water-insoluble filleris microcrystalline cellulose; the binder is hydroxypropylmethylcellulose; and the lubricant is glyceryl behenate.

In another specific embodiment, the pharmaceutical formula comprisesbetween 12 wt % to 20 wt % water insoluble filler, between 34 wt/o to 46wt %, water soluble filler, between 35 wt % and 40 wt % Formula (I)Hemitartrate, between 2 wt/o and 6 wt % binder and between 0.1 wt % and2 wt % binder, all on a dry solids basis. More specifically, thewater-soluble filler is lactose monohydrate; and the water-insolublefiller is microcrystalline cellulose. Even more specifically thewater-soluble filler is lactose monohydrate; the water-insoluble filleris microcrystalline cellulose; the binder is hydroxypropylmethylcellulose; and the lubricant is glyceryl behenate.

In another specific embodiment, the pharmaceutical formula comprisesbetween 14 wt % to 18 wt % water insoluble filler, between 38 wt/o to 44wt %, water soluble filler, between 35 wt % and 40 wt % Formula (I)Hemitartrate, between 2 wt % and 6 wt % binder and between 0.1 wt % and2 wt % binder, all on a dry solids basis. More specifically, thewater-soluble filler is lactose monohydrate; and the water-insolublefiller is microcrystalline cellulose. Even more specifically thewater-soluble filler is lactose monohydrate; the water-insoluble filleris microcrystalline cellulose; the binder is hydroxypropylmethylcellulose; and the lubricant is glyceryl behenate.

In another specific embodiment, the pharmaceutical formula comprisesbetween 14 wt % to 18 wt % water insoluble filler, between 38 wt % to 44wt/o, water soluble filler, between 36 wt/o and 39 wt/o Formula (I)Hemitartrate, between 2 wt % and 6 wt % binder and between 0.1 wt % and2 wt % binder, all on a dry solids basis. More specifically, thewater-soluble filler is lactose monohydrate; and the water-insolublefiller is microcrystalline cellulose. Even more specifically thewater-soluble filler is lactose monohydrate; the water-insoluble filleris microcrystalline cellulose; the binder is hydroxypropylmethylcellulose; and the lubricant is glyceryl behenate.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

EXPERIMENTAL Example 1: Preparation of Salts of Formula (I)

The hemitartrate salt of Formula I is readily crystallized and exhibitsmany beneficial properties as compared to other salts. For example, thefollowing acids were used in the preparation of salts of the compoundrepresented by Formula (I): citric acid (generating salts in 1:1, 1:2,and 1:3 (salt:Formula I) ratios); L-malic (1:1 and 1:2); methanesulfonic acid (1:1); fumaric acid (1:1 and 1:2); hydrochloric acid(1:1); acetic acid (1:1) and tartaric acid (1:1 and 1:2). Only saltsgenerated by hydrochloric acid (1:1); tartaric acid (1:1) and tartaricacid (1:2) were of solid form. Of these three salts hydrochloric acid(1:1) and tartaric acid (1:1) were found to be hygroscopic andnon-crystalline and therefore unacceptable for use in a pharmaceuticalproduct. The hemitartrate (1 salt: 2 Formula I) of the compoundrepresented by Formula I was found to be crystalline andnon-hygroscopic.

Acetone Preparation of Formula (I) Hemitartrate

L-tartaric acid (6.02 g, 40.11 mmol, 0.497 equivalents) was dissolved inacetone (175 mL) by refluxing the solution and then cooling to roomtemperature. Formula (I) Free Base (32.67 g, 80.76 mmol) was dissolvedin acetone (300 mL) at room temperature. The L-tartaric acid solutionwas added to the Formula (I) Free Base solution at room temperature over15 min. A white precipitate formed half way through the addition. Themixture was stirred at room temperature for 0.5 h hours and then brieflyrefluxed and cooled to room temperature. After stirring a roomtemperature for 0.5 h, the white precipitate was filtered. The whitesolid was washed twice with acetone (2×130 mL). The solid was air driedand then vacuum dried at 55-60° C. The yield was 36.66 g (95%).

5% Methanol in Acetone Preparation of Formula (I) Hemitartrate.

Formula (I) Free Base, 10 g/24.7 mmol, was dissolved in 5%Methanol/Acetone 120 mL or 240 mL. L-tartaric acid, 1.85 g/12.3 mmol,was dissolved in 5% Methanol/Acetone 60 mL or 120 mL (N or 2N) bywarming to 40-45° C., and this solution was added to the first solution.After 1 hour without precipitation, 1 mg of Formula (I) Hemitartrate wasadded as a seed crystal. Precipitation occurred after 5 minutes, and thereaction continued to stir for 30 minutes more. The reaction was thenheated at reflux for 5 minutes (the precipitate was completely soluble)and then cooled to room temperature in a water bath 20-22° C.Precipitate formed and the reaction continued to stir for 3 hours. Thefinal product was collected by filtration and was washed with acetone,2×40 mL, and then dried in the vacuum oven at 55-60° C. for 16 hours.Product weight was 8.72 g/74% yield.

1% Water in Acetone Preparation of Formula (I) Hemitartrate.

Formula (I) Free Base (10 g/24.7 mmol) was dissolved in 1% Water/Acetone120 mL or 240 mL at room temperature. L-tartaric acid, 1.85 g/12.3 mmol,was dissolved in 1% Water/Acetone 60 mL or 120 mL (N or 2N) by warmingto 40-45° C., and this solution was added to the first solution. After 1hour without precipitation, 1 mg of Formula (I) hemitartrate was addedas a seed crystal. Precipitation occurred after 5 minutes, and thereaction continued to stir for 30 minutes. The reaction was then heatedat reflux for 5 minutes (the precipitate was not completely soluble) andthen cooled to room temperature in a water bath 20-22° C. Precipitateformed and the reaction continued to stir for 3 hours. The final productwas collected by filtration and was washed with acetone, 2×40 mL, andthen dried in the vacuum oven at 55-60° C. for 16 hours. Product weightwas 8.62 g 73% yield.

5% Methanol in Acetone Recrystallization of Formula (I) Hemitartrate.

Formula (I) Hemitartrate (3.06 g) was dissolved in 116 mL of 5% methanolin acetone at reflux. The solution was cooled to room temperature andstirred at room temperature for 2 h. The white precipitate was filteredand washed with 10 mL of 5% methanol in acetone and then acetone (15mL). After vacuum drying for 18 h at 55-60° C., received 2.38 g ofFormula (I) Hemitartrate (78% recovery).

1% H₂O in Acetone Recrystallization of Formula (I) Hemitartrate.

Formula (I) Hemitartrate (3.05 g) was dissolved in 125 mL of 1% H₂O inacetone at reflux. The solution was cooled to room temperature andstirred at room temperature for 2 h. The white precipitate was filteredand washed with 10 mL of 1% H₂O in acetone and then acetone (15 mL).After vacuum drying overnight at 55-60° C., 2.35 g of Formula (I)Hemitartrate (77% recovery) was obtained.

Example 2: Preparation Crystalline Formula (I) Hemitartrate

Formula (I) Hemitartrate was crystallized by several methods. Batch 1was prepared using ethyl acetate/acetone solvents and dried at roomtemperature. Batch 3 was prepared using ethyl acetate/acetone solventsand recrystallized from ethyl acetate. Batch 4 was recrystallized fromacetone using Batch 1 material. Batch 5 was recrystallized fromisopropanol. Batch 7 was prepared using ethyl acetate/acetone solventsimilar to Batch 1 but in a large scale, Batch 8 was prepared usingacetone only with no further recrystallization. Batch 9 was preparedusing acetone only with brief reflux, again no furtherrecrystallization.

TABLE 1 Summary of polymorphism screening of Batches 1-9 of Formula (I)Hemitartrate DSC Batch Melting Pont (° C.) No. Processing MethodEnthalpy (J/g) Microscope TGA 1 Acetone/ethyl acetate 162 −81.4 Clystal99.91% at 100° C. precipitation* 98.73% at 175° C. 2 Acetone/ethylacetate 164 −95.6 Crystal N/A precipitation-dried at room-temperature* 3Acetone/ethyl acetate 166 −97.8 Crystal 100.0% at 100° C. precipitation-dried at 99.98% at 153° C. 55-60° C. 4 Recrystallization 166 −107.2Crystal 100.2% at 100° C. from acetone 100.2% at 153° C. 5Recrystallization 166 −102.6 Crystal 100.0% at 100° C. from isopropanol100.0% at 153° C. 7 Acetone/ethyl acetate 166 −99.4 Crystal** 100.1% at100° C. precipitation 99.91% at 153° C. 8 Acetone precipitation 165−100.7 Crystal** 100.0% at 100° C. 100.0% at 153° C. 9 Acetoneprecipitation 165 −100.2 Crystal** with brief reflux *containing somefree base in the DSC thermogram. **containing habits changed in thesebatches from rod, plate-shaped to needle, rod, and irregular shapes.

Crystal forms of Formula (I) Hemitartrate were also prepared using slowevaporation, slow cooling, fast cooling and anti-solvent precipitationwith a variety of solvents.

Slow Evaporation Method. A weighed sample (usually 20 mg) was treatedwith aliquots of the test solvent. Aliquots were typically 100-200 μL.Between solvent additions, the mixture was shaken or sonicated. When thesolids dissolved, as judged by visual inspection, the solution wasallowed to evaporate under ambient conditions in an open vial coveredwith aluminum foil perforated with pinholes. Solubilities were estimatedfrom these experiments based on the total solvent added to obtain aclear solution.

TABLE 2 Approximate solubility of Formula (I) Hemitartrate at roomtemperature (20-25° C.). Approximate Organic Solvent Solubility (mg/mL)Heptane Not Available Hexane Not Available Toluene <5 Dichloromethane100 Ethanol 29 Isopropyl alcohol <5 Acetonitrile <5 Ethyl Acetate <5Methanol >200 Acetone <5 Methyl t-butyl ether (TBME) <5 p-Dioxane <5Tetrahydrofuran (THF) <5

TABLE 3 Summary of polymorphism using slow evaporation approach. Solidform DSC generated Melting Organic from Slow Pont (° C.) SolventEvaporation Enthalpy (J/g) Microscope TGA Methanol No N/A N/A N/A N/AEthanol Yes 165 −95.0 Crystal** 100.0% at 100° C. 100.0% at 150° C.**particles were plate and rod-shaped

Slow/Fast Cooling Method. Formula (I) Hemitartrate was dissolved in atest solvent at 50-60° C. The resulting solution was then allowed tocool to ambient temperature (slow cool). If no solids formed after aday, the vials were placed in a refrigerator. For fast cool experiments,the resulting solution was then allowed to cool in a refrigerator. Thesolids were collected by filtration an air-dried.

TABLE 4 Summary of polymorphism using slow cooling approach. Solid formDSC generated Melting Organic from Slow Pont (° C.) Solvent CoolingEnthalpy (J/g) Microscope TGA Ethanol Yes 167 −106.2 Crystal** 100.1% at100° C. 100.1% at 150° C. **particles were plate and rod-shaped

TABLE 5 Summary of polymorphism using fast cooling approach. Solid formDSC generated Melting Organic from Fast Pont (° C.) Solvent CoolingEnthalpy (J/g) Microscope TGA Ethanol Yes 167 −106.2 Crystal** 100.0% at100° C. 100.0% at 150° C. **particles were plate and rod-shaped

Anti-Solvent Method. Formula (I) Hemitartrate was dissolved in asolvent. An anti-solvent was added to the solution. The solids thatformed were collected by filtration an air-dried.

TABLE 6 Summary of polymorphism screening using anti-solvent approachSolid form generated DSC from Anti- Melting Organic solvent Pont (° C.)Micro- Solvent Approach Enthalpy (J/g) scope TGA Methanol/ Yes 167 −99.5Crystal* 100.1% at 100° C. ethyl 100.1% at 150° C. acetate Methanol/ Yes167 −106.2 Crystal* 100.3% at 100° C. acetone 100.2% at 150° C.Methanol/ No N/A N/A N/A N/A acetonitrile Methanol/ No N/A N/A N/A N/Atoluene Methanol/ No N/A N/A N/A N/A THF Methanol/ Yes 167 −102.0 Crystal* 100.2% at 100° C. TBME 100.1% at 150° C. Methanol/ No N/A N/AN/A N/A p-dioxane Water/THF No N/A N/A N/A N/A Water/ No N/A N/A N/A N/ATMBE Water/ No N/A N/A N/A N/A isopropanol Water/ No N/A N/A N/A N/Aacetonitrile Water/ No N/A N/A N/A N/A acetone Dicholoro- Yes 165 −89.2Crystal** 100.0% at 100° C. methane/ 99.99% at 150° C. hepaticDicholoro- Yes 167 −97.8 Crystal* 100.2% at 100° C. methane/ 100.1% at150° C. ethyl acetate Dicholoro- Yes 164 −89.8 Crystal* 99.95% at 100°C. methane/ 99.86% at 150° C. toluene Dicholoro- Yes 167 −98.6 Crystal**100.0% at 100° C. methane/ 99.91% at 150° C. TBME Dicholoro- Yes N/A N/AN/A N/A methane/ p- (little) dioxane Dicholoro- No N/A N/A N/A N/Amethane/ isopropanol The particles were plate and rod-shaped.**Individual particles had more than one bifrigence color. ***Theparticles were needle and rod-shaped.

Example 3: Physical Properties of Formula (I) Hemitartrate

Differential scanning calorimetry (DSC). DSC data was collected on a TAQ100 instrument utilizing nitrogen as the purge gas. Approximately 2-5mg of sample was weighed accurately into an aluminum DSC pan. The panwas covered with a lid and perforated with a forceps. The sample cellwas equilibrated at 30° C. and heated at a rate of 10° C. per minute toa final temperature of 220° C.

Hot stage microscopy. Hot stage microscopy was performed using a Linkamhot stage (model FTIR 600) mounted on a Leica DM LP microscope equippedwith a Sony DXC-970MD 3CCD camera for image collection. A 40× objectivewas used with polarized light to view samples. Each samples was placedbetween two cover slips. Each sample was visually observed as the stagewas heated. Images were captured using Links version 2.27 (Linkam). Thehot stage was calibrated using USP melting point standards.

The endothermic transition observed in the DSC profile was confirmed tobe a melting transition at a temperature between 160-163° C. by hotstage microscopy.

Example 4: X-Ray Powder Diffraction of Formula (I) Hemitartrate

All the X-ray Powder Diffraction (XRPD) analyses were done at SSCI, Inc.(West Lafayette, Ind. 47906). XPRD analyses were performed using aShimadzu XRD-6000 X-ray powder diffractometer using Cu K α radiation.The instrument is equipped with a fine focus X-ray tube. The tubevoltage and amperage were set to 40 kV and 40 mA, respectively. Thedivergence and scattering slits were set at 1° and the receiving slitwas set at 0.15 mm. Diffracted radiation was detected by a NaIscintillation detector. The theta-two theta continuous scan at 3°/min(0.4 sec/0.02° step) from 2.5 to 40° 20 was used. A silicon standard wasanalyzed to check the instrument alignment. Data were collected andanalyzed using XRD-6000 v 4.1.

Example 5: Comparison of Formula (I) Hemitartrate to Formula (I) FreeBase

The solid characterization of the free base and the hemitartrate saltare summarized in Table 7. Formula I Hemitartrate has superiorproperties as compared to Formula I free base. For example, Formula IHemitartrate has a higher melting point (>150° C.), higher packingenergy (greater endothermic enthalpy), lower variance in particle size,higher aqueous solubility (over 300 mg/mL in water), suitable crystalshape, and higher bulk density as compared to Formula I Free Base.

TABLE 7 Summary of solid state and physical and chemical properties ofFormula (1) Free Base and Formula (1) Hemitartrate. Formula (I) FreeFormula (I) Physical Characteristics Base Hemitartrate Melting Point (°C.) 86-88 163 Endothermic enthalpy (J/g) 75-82 96-106 Particle size (μm)<10 to 100 ~3 (Average) Aqueous solubility (mg/mL) 0.04 >216 CrystallineYes Yes Crystal Shape Needle Plate, rod, some irregular Hygroscopicity(40° C./75% None None RH) Bulk Density ~0.2 0.4-0.5

Example 6: In Vitro Activity and Specificity

Activity of Formula (I) Hemitartrate at inhibiting glycosphingolipidsynthesis in vitro. Two assays were used to quantify the inhibitoryactivity of Formula (I) Hemitartrate for glucosylceramide synthase.Since glucosylceramide is the first and rate-limiting step in thebiosynthesis of glycosphingolipids, a flow cytometry assay that measuredcell surface levels of GM 1 and GM3 was used to indirectly assess theactivity of the inhibitor in intact cells. Incubating K562 or B16/F10cells for 72 h with increasing amounts of Formula (I) Hemitartrate(0.6-1000 nM) resulted in a dose-dependent reduction of cell surfacelevels of both GM1 and GM3. The mean IC₅₀ value for inhibiting the cellsurface presentation of GM1 in K562 cells was 24 nM (range 14-34 nM)(Table 8) and that for GM3 in B16/F10 cells was 29 nM (range 12-48 nM).No overt cellular toxicity was noted in either cell line even whentested at the highest dose.

An alternative assay for activity measured inhibition ofglucosylceramide synthase in human cell derived microsomes. In thisassay, microsomes were prepared from human melanoma A375 cells bysonication and centrifugation. The microsomal preparation was incubatedwith a fluorescent ceramide substrate (NBD-C6-ceramide), UDP-glucose,and increasing amounts of Formula (I) Hemitartrate (0-1000 nM) for onehour at room temperature. Following the incubation, fluorescentlylabeled glucosylceramide and unreacted ceramide were separated andquantitated by reverse-phase HPLC and fluorescence detection. In thisassay the IC₅₀ value for inhibiting glucosylceramide synthesis rangedfrom 20 to 40 nM. This value was similar to those obtained above for GM1and GM3 and suggests that measurements of these cell surface glycolipidsare good surrogates of the activity of Formula (I) Hemitartrate forglucosylceramide synthase.

Specificity of substrate synthesis inhibition by Formula (I)Hemitartrate. The specificity of Formula (I) Hemitartrate was evaluatedin a series of in vitro cell-based and cell-free assays. The intestinalglycosidase enzymes were assayed in rat tissue homogenates (see U.Andersson, et al., Biochem. Pharm. 59 (2000) 821-829, the entireteachings of which are incorporated herein by reference), and theglycogen debranching enzyme was assayed in a cell free assay asdescribed (see U. Andersson, et al., Biochem. Pharm. 67 (2004) 697-705,the entire teachings of which are incorporated herein by reference). Nodetectable inhibition of intestinal glycosidases (lactase, maltase,sucrase), α-glucosidase I and II, and the cytosolic debranching enzyme(α-1,6-glucosidase), was found at concentrations up to 2500 μM (Table8).

Non-lysosomal glucosylceramidase and lysosomal glucocerebrosidase wereassayed in intact human cells using C₆-NBD-glucosylceramide as substrate(see H. S. Overkleeft, et al. J. Biol. Chem. 273 (1998) 26522-26527, theentire teachings of which are incorporated herein by reference).Conduritol β epoxide (a specific inhibitor of lysosomalglucocerebrosidase) was used to differentiate lysosomal versus thenon-lysosomal activity. Glucocerebrosidase activity was also measured byfluorescence-activated cell sorting (FACS). K562 cells were culturedwith increasing amounts of Formula (I) Hemitartrate in the presence of 1μM 5-(pentafluorobenzoylamino)-fluorescein di-P-D-glucopyranoside(PFB-FDGlu, Molecular Probes/Invitrogen. Carlsbad, Calif.) for 30-60min. Cells were immediately chilled on ice and the fluorescencequantitated is above. The non-lysosomal glucosylceramidase was weaklyinhibited with an IC₅₀ of 1600 μM. There was no inhibition of lysosomalglucocerebrosidase, the enzyme that is deficient in Gaucher disease, upto the highest concentration of 2500 μM (Table 8). Hence, a differentialof approximately 40,000 in the concentration was required to inhibitglucosylceramide synthase compared to any of the other enzymes tested.

TABLE 8 Biochemical activities Formula (I) Hemitartrate in vitroSubstrate inhibition potency (in vitro IC₅₀): ~0.024 μM Enzymespecificities, IC₅₀: α-Glucosidase I and II: >2500 μM Lysosomalglucocerebrosidase (GBA1): >2500 μM μM Non-lysosomal glucosylceramidase1600 μM (GBA2): Glycogen debranching enzyme: >2500 μM Enzymespecificities, K_(i): Sucrase inhibition: No inhib. to 10 μM Maltaseinhibition: No inhib. to 10 μM

Example 7: Improved Management of Lysosomal Glucosylceramide Levels in aMouse Model A. Fabry Disease.

To determine if the combined use of both enzyme replacement therapy(ERT) and substrate reduction therapy (SRT) may maintain enzymedebulking or provide additional benefits, the relative efficacies ofseparate and combined therapies in a murine model of Fabry disease(Fabry-Rag) were compared. The parental Fabry mice is described in Wang,A M et al. Am. J. Hum. Genet. 59: A208 (1996). The Fabry-Rag is crossedwith a RAG-1 mouse and does not develop mature lymphocytes or T-cells(immune-compromised).

Animal Studies.

For the monotherapy studies, Fabry mice were put on study at 1 month old(prevention model). Treatment groups received Formula (I) Hemitartrate(Genzyme Corp., Cambridge, Mass.) as a component of the pellet fooddiet. The drug was formulated at 0.15% (w/w) in standard 5053 mouse chow(TestDiet, Richmond, Ind.) and provided ad libitum. This formulationprovided 300 mg/kg of Formula (I) Hemitartrate per day in a 25 g mouse.

For the combination therapy studies, Fabry-Rag mice were put on study at3 months old (treatment model). Mice in group A received intravenousinjections of recombinant human alpha-galactosidase A (Genzyme Corp.) ata dose of 1 mg/kg every 2 months (i.e. 3, 5, 7 and 9 months old). GroupB received the same intravenous enzyme doses plus it received Formula(I) Hemitartrate (Genzyme Corp., Cambridge, Mass.) as a component of thepellet food diet. The drug was formulated at 0.15% (w/w) in standard5053 mouse chow (TestDiet, Richmond, Ind.) and provided ad libitum. Thisformulation provided 300 mg/kg of Formula (I) Hemitartrate per day in a25 g mouse. Group C received enzyme injections every 4 months (i.e. 3and 7 months old) and was on the same drug-in-food diet as group B.Group D received only the drug-in-food diet (same as groups B and C).Group E was untreated Fabry-Rag mice and group F were wild-typecontrols. See FIG. 10.

Quantitation of Tissue Globotriaosylceramide (GL-3, GH3) Levels

Quantitation of GL-3 was by Tandem Mass Spectrometry essentially as forGL-1.

Hot plate Assay was performed as described previously (Ziegler, R J etal. Molec. Ther. 15(3), 492-500 (2007).

Results

Monotherapy of Fabry Mice with Formula (I) Hemitartrate

SRT was evaluated in a mouse model of Fabry disease, which is caused bya deficiency of a-galactosidase A activity. Therapy with Formula (I)Hemitartrate started with one month-old Fabry mice and continued untilthe mice reached one year of age. The animals were dosed with 300 mg/kgFormula (I) Hemitartrate in their diet each day. Behavioral tests (i.e.,hot-plate assay) and biochemical tests (i.e., urinalysis and GL-3 levelanalysis in tissues/blood/urine) of the mice were performed bimonthly.

As shown in FIG. 7, administration of Formula (I) Hemitartrate toFabry-Rag mice over a period of 11 months abated the rate of lysosomalaccumulation of globotriaoslyceramide (GL-3) in the somatic organs(liver, kidney, heart and spleen) by approximately 50%. This translatedto a delay in disease progression as evidenced by a later presentationof insensitivity to an aversive heat stimulus (see FIG. 8) and aprevention of deterioration of urinalysis factors, e.g., urine volume,creatinine and sodium levels (see FIG. 9). Hence, Formula (T)Hemitartrate-mediated inhibition of glucosylceramide synthase thatcatalyzes the first step in the synthesis of glycosphingolipids, is notonly advantageous in animal models of Gaucher disease but also of Fabrydisease, and could also have positive effects in otherglycosphingolipidoses.

Combination Therapy of Fabry Mice with α-Galactosidase A and Formula (I)Hemitartrate

The efficacy of ERT alone and in combination with SRT using Formula (I)Hemitartrate was evaluated in five populations of Fabry-Rag mice(n=12/group). Beginning at three-months of age, the mice were subjectedto a schedule of behavioral tests (i.e. hot-plate assay) and biochemicaltests (i.e., GL-3 level analysis in tissues/blood/urine), as shown inFIG. 10. In mice subjected to ERT, 1 mg/kg doses of a-galactosidase Awere administered on the schedule as shown in FIG. 10. In mice subjectedto SRT, 300 mg/kg doses of Formula (I) Hemitartrate were administereddaily in the mouse diet.

As shown in FIG. 11, ERT reduces blood GL-3 levels in Fabry-Rag mice,whereas SRT does not. As shown in FIG. 12, combination ERT/SRT is mosteffective at reducing GL-3 levels in Fabry-Rag mice liver and kidney.

As shown in FIG. 13, SRT reduces urine GL-3 levels in Fabry-Rag mice,whereas ERT does not. As shown in FIG. 14, SRT but not ERT delays onsetof heat-insensitivity in Fabry-Rag mice.

In summary, Fabry-Rag mice treated with a combination Fabrazyme andFormula (I) Hemitartrate exhibited improvements in disease markers overERT or SRT alone in a treatment model in the following ways:significantly reduced liver and kidney GL-3 accumulation withcombination therapy; improved urine GL-3 in SRT groups; improved bloodGL-3 in ERT groups; and delayed peripheral neuropathy in SRT groups.

B. Gaucher disease. To determine if the sequential use of both enzymereplacement therapy (ERT) and substrate reduction therapy (SRT) mayprovide additional benefits, the relative efficacies of separate andsequential therapies in a murine model of Gaucher disease (D409V/null)were compared.

Methods

Animal studies. Procedures involving animals were reviewed and approvedby the Institutional Animal Care and Use Committee (IACUC) at GenzymeCorporation following the guidelines issued by the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC). TheGaucher mouse (D409V/null) is a model of type 1 Gaucher diseaseexhibiting accumulation of glucosylceramide in liver, spleen and lungsbut lacks bone or brain pathology (see Y-H. Xu, et al., Am. J. Pathol.163, 2003, 2093-2101, the entire teachings of which are incorporatedherein by reference). Animals of both sexes were placed on study at 3months of age as previous experiments had indicated that there was nodifference in response between males and females to recombinantglucocerebrosidase or Formula (I) Hemitartrate. The study had 6 groupsof mice with group A being sacrificed after 2 weeks to provide baselinelevels of tissue glucosylceramide. Groups B, C, and D all receivedrecombinant human glucocerebrosidase (Genzyme Corp., Cambridge, Mass.)(10 mg/kg) intravenously via a tail-vein (100 μL) every 2 days for atotal of 8 injections. Group B was sacrificed at the end of this regimen(at the same time as group A) to provide enzyme-reduced levels of tissueglucosylceramide. Groups D and E were both fed Formula (I) Hemitartrate(Genzyme Corp., Cambridge, Mass.) as a component of the pellet fooddiet. The drug was formulated at 0.075% (w/w) in standard 5053 mousechow (TestDiet, Richmond, Ind.) and provided ad libitum. Thisformulation provided 150 mg/kg of Formula (I) Hemitartrate per day in a25 g mouse. Group F received no treatment and was sacrificed along withgroups C, D and E 12 weeks after the start of the study. Foodconsumption and mouse weights were monitored three times per week todetermine drug intake and the potential impact of the drug on overallhealth. Animals were killed by carbon dioxide inhalation and theirtissues harvested immediately. Half of each tissue was snap frozen ondry ice and stored at −80° C. until ready for further processing. Theother half was processed for histological analysis.

Quantitation of tissue glucosylceramide levels Glucosylceramide levelswere quantified by mass spectrometry as described previously (see K.McEachern, et al., J. Gene. Med. 8 (2006) 719-729; T. Doering, J. Biol.Chem. 274 (1999) 11038-11045, the entire teachings of both areincorporated herein by reference). A known mass of tissue washomogenized in 2:1 (v/v) chloroform:methanol and incubated at 37° C. for15 min. Samples were centrifuged and the supernatants were extractedwith 0.2 volumes of water overnight at 4° C. The samples werecentrifuged, the aqueous phase was discarded, and the organic phase wasdried down to a film under nitrogen. For electrospray ionization massspectrometry (ESI/MS) analysis, tissue samples were reconstituted to theequivalent of 50 ng original tissue weight in 1 ml chloroform:methanol(2:1, v/v) and vortexed for 5 min. Aliquots (40 μL) of each sample weredelivered to Waters total recovery vials and 50 μL of a 10 μg/mLd3-C16-GL-1 internal standard (Matreya, Inc., Pleasant Gap, Pa.) wasadded. Samples were dried under nitrogen and reconstituted with 200 μLof 1:4 (v/v) DMSO:methanol. ESI/MS analysis of glucosylceramides ofdifferent carbon chain lengths was performed on a Waters alliance HPLC(Separation Module 2695) coupled to a Micromass Quattro Micro systemequipped with an electrospray ion source. Lipid extract samples (20 μL)were injected onto a C8 column (4 mL×3 mm i.d; Phenomenex, Torrance,Calif.) at 45° C. and eluted with a gradient of 50 to 100% acetonitrile(2 mM ammonium acetate, 0.1% formic acid) at 0.5 mL/min. The first 0.5min was held at 50% organic and then quickly switched to 100% for thefinal 3.5 min. The source temperature was held constant at 150° C. andnitrogen was used as the desolvation gas at a flow rate of 670 L/h. Thecapillary voltage was maintained at 3.80 KV with a cone voltage of 23 V,while the dwell time for each ion species was 100 ms. Spectra wereacquired by the MRM mode to monitor eight dominant isoforms (C16:0,C18:0, C20:0, C22:1, C22:0, C22:1-OH, C24:1, and C24:0). Quantitation ofglucosylceramide was based on the sum of these eight isoforms relativeto the internal standard, with a calibration curve ranging from 0.1 to10 μg/mL.

Histology. For histological analysis, tissues were fixed in zincformalin (Electron Microscopy Sciences, Hatfield, Pa.) at roomtemperature for 24 h, then stored in PBS at 4° C. until ready forfurther processing. All samples were dehydrated in ethanol, cleared inxylenes, infiltrated and embedded in Surgipath R paraffin (Surgipath,Richmond, Ill.). Five micron sections were cut using a rotary microtomeand dried in a 60° C. oven prior to staining. Sections weredeparaffinized in Hemo-De (Scientific Safety Solvents, Keller, Tex.) andrehydrated in descending concentrations of ethanol followed by a PBSwash. The sections were stained with Hematoxylin and Eosin (H&E) andlabeled using a rat anti-mouse CD68 monoclonal antibody (Serotec,Raleigh, N.C.) to identify macrophages. After washing for 5 min in PBS,the slides were dehydrated in ethanol and cleared in Hemo-De prior tomounting with SHUR/Mount™ coverglass mounting medium (TBS, Durham,N.C.). The percent area of CD68 immunopositivity in the liver wasquantified using MetaMorph (MDS Analytical Technologies, Toronto,Canada) analysis of ten 400× images per tissue section. A boardcertified veterinary pathologist blinded to group designation examinedall the sections.

Results

Dosing regimen of glucocerebrosidase for debulking accumulated GL1 inthe liver, spleen and lung of 3 month-old Gaucher mice. To investigatethe relative merits of combination and monotherapy with either enzyme orsubstrate reduction therapy, the enzyme regimen that maximally depletedGL1 levels in the visceral organs of Gaucher mice was first determined.Three month-old Gaucher mice (D409V/null) were intravenouslyadministered 2, 4 or 8 doses of 10 mg/kg recombinant humanglucocerebrosidase. The mice that were treated with 2 or 4 doses of theenzyme received drug infusions every 3 days while those that weretreated with 8 doses received the enzyme every 2 days. The use of ashorter time interval between infusions in animals that received 8treatments was designed to minimize the potential impact of any immuneresponse to the administered human enzyme. The animals were killed 7days following the last enzyme infusion and the amount of GL1 remainingin their livers, spleens, and lungs were measured.

Treatment with 2 doses of glucocerebrosidase reduced the levels of GL1in the liver by 50%. Increasing the number of enzyme infusions to 4 or8, as expected, reduced the liver GL1 levels to a greater extent (byapproximately 75%). The less than complete lowering of GL1 levels, evenwith 8 doses, is consistent with the experience in Gaucher subjectsshowing that hepatosplenomegaly is reduced only after an extended periodof treatment (see G. A. Grabowski, et al., Ann. Int. Med. 122 (1995)33-39, the entire teachings of which are incorporated herein byreference). The substrate levels in the spleens of Gaucher mice weremore refractory to enzyme treatment. Administration of 2 doses ofglucocerebrosidase did not significantly alter GL1 levels from thosenoted in untreated controls. Increasing the number of enzyme infusionsto 4 or 8 reduced the splenic GL1 levels by about 50%. In the lung, areduction to approximately 60% of untreated control was observed after 8doses. The slightly lower extent of substrate reduction in the lung wasprobably due to poorer accessibility of the infused enzyme to thelipid-laden alveolar macrophages. The observation of greater GL1clearance in the liver when compared with the spleen and lung likelyreflects the biodistribution of the enzyme following systemic infusion(see S. M. Van Patten, et al. Glycobiology 17 (2007) 467-478, the entireteachings of which are incorporated by reference). Based on theseresults, the treatment regimen consisting of 8 consecutive doses of 10mg/kg glucocerebrosidase administered at 2 days intervals was used forthe subsequent studies.

Relative abilities of enzyme and substrate reduction therapy to lowerGL1 levels in the liver of Gaucher mice. Cohorts of 3-month-old Gauchermice were treated with either recombinant glucocerebrosidase or Formula(I) Hemitartrate separately or sequentially. Mice in groups B, C and Dwere given 8 doses of enzyme as described above (over a period of 2weeks) to clear accumulated GL1. Different groups were then fed eitherregular chow or chow containing Formula (I) Hemitartrate (150 mg/kg/day)for an additional 10 weeks with group F receiving no treatment andserving as the naïve control. Irrespective of the chow formulation, themice ate comparable amounts of food and there were no discernibledifferences in weight gain. Approximately 80% of the stored GL1 levelswere cleared from the liver following 2 weeks of enzyme therapy alone.When these animals were allowed to progress without further treatmentfor 10 weeks, their liver GL1 levels increased indicating thatre-accumulation of the substrate had occurred during the interveningperiod (FIG. 2, column C). These levels were not significantly differentfrom those of untreated controls (FIG. 2, column F). However, if themice were treated with enzyme and then Formula (I) Hemitartrate in theirfood over a 10 week period, their liver GL1 levels were significantlylower than the untreated controls (FIG. 2, column D & F). This resultsuggests that the additional treatment with Formula (I) Hemitartrate hadslowed the re-accumulation of the substrate. Interestingly, Gaucher micetreated with Formula (I) Hemitartrate alone during the entire studyperiod (12 weeks) also showed lower GL-1 levels (FIG. 2, column E) whencompared to untreated, age-matched controls (FIG. 2, column F) thoughthe difference was not significant. The ability of SRT alone to reduceGL1 levels in this animal model is consistent with our previous report(see K. A. McEachern, et al., Mol. Genet. Metab. 91 (2007) 259-267, theentire teachings of which are incorporated herein by reference) andlikely reflects the fact that the Gaucher mice (D409V/null) retainresidual enzymatic activity (see Y-H. Xu, et al., Am. J. Pathol. 163,2003, 2093-2101, the entire teachings of which are incorporated hereinby reference).

Relative abilities of enzyme and substrate reduction therapy to lowerGL1 levels in the spleen of Gaucher mice. Treating 3 month-old Gauchermice with recombinant glucocerebrosidase alone for 2 weeks reducedsplenic GL1 levels by approximately 60% (FIG. 3, column B). When theseanimals were allowed to age for an additional 10 weeks without furtherintervention, the substrate levels returned to those observed at thestart of the study (FIG. 3, column C) and were not significantlydifferent from the untreated control (FIG. 3, column F). This suggeststhat the rate of re-accumulation of GL1 in the spleen was higher than inthe liver. This supposition was also supported by the observation ofhigher basal levels of the substrate in the spleen (˜1500 mg/g tissue;FIG. 2, column A) than in the liver (˜500 mg/g tissue; FIG. 3, columnA). Animals that had been treated with enzyme and then Formula (I)Hemitartrate for the next 10 weeks showed the greatest reduction insplenic GL1 levels (FIG. 3, column D) and these were significantly lowerthan those in the untreated control spleens (FIG. 3, column F). Thisindicated that the deployment of SRT not only delayed there-accumulation of substrate but also acted to further reduce the burdenof storage in this organ. It would appear that at least in thisinstance, the net effect of the residual endogenous enzyme and substratereduction led to a further decline in overall substrate levels. Theobservation of lower splenic GL1 levels in the mice treated with Formula(I) Hemitartrate alone for 12 weeks (FIG. 3, column E) than in untreatedcontrols (FIG. 3, column F) is consistent with this notion, though thedifference was not significant. Hence, in mild Gaucher type 1 patientswith high residual enzyme activity, treatment with ERT followed by SRTcould potentially accelerate the rate and perhaps even the extent ofclearance of the offending substrate.

Relative abilities of enzyme and substrate reduction therapy to lowerGL1 levels in the lung of Gaucher mice. As noted earlier, pulmonary GL1levels were least effectively cleared by intravenous administration ofrecombinant glucocerebrosidase. Treatment of 3 month-old Gaucher micewith enzyme for 2 weeks resulted in only a 30% reduction in substratelevels in the lung (FIG. 4, column B). The cohort of animals fed normalchow for the next ensuing 10 weeks showed, as expected, re-accumulationof GL1 and were not significantly different from the untreated levels(FIG. 4, column C & F). In contrast, animals fed chow containing Formula(I) Hemitartrate over the same intervening period showed a reduction insubstrate levels to below those administered enzyme alone (FIG. 4,column D) and were significantly lower than those in the untreatedcontrols (FIG. 4, column F). Again, this suggests that in the lung, asin the spleen, the net effect of Formula (I) Hemitartrate (in thepresence of residual endogenous enzyme activity) not only retarded there-accumulation of GL1 but also acted to further reduce them to belowthe starting levels. As with the other visceral organs, treatment byFormula (I) Hemitartrate alone was effective in lowering pulmonary GL1levels (FIG. 4, column E) when compared to untreated controls (FIG. 4,column F).

Histopathological analysis of the liver of Gaucher mice after enzyme andsubstrate reduction treatment. To visualize the effects of the differenttherapeutic regimens in the liver, tissue sections were stained forCD68, a macrophage marker. Analysis of liver sections from untreated 3month-old Gaucher mice showed the presence of large numbers oflipid-engorged, CD68-positive Gaucher cells that remained largelyunchanged when analyzed 12 weeks later. Consistent with the biochemicaldata above, livers of animals administered recombinantglucocerebrosidase over a period of 2 weeks showed substantial clearanceof the lipid in these abnormal macrophages. If these animals wereallowed to age an additional 10 weeks without further treatment, therewas evidence of re-accumulation of GL1 as indicated by the re-emergenceof Gaucher cells. However, this increase in Gaucher cells was negated ifthe mice were given substrate reduction therapy with Formula (I)Hemitartrate over the same intervening period. As noted earlier, Gauchermice that received Formula (I) Hemitartrate alone also showed reducedaccumulation of the substrate, although not to the same degree as thosethat received a combination of ERT and SRT. The extent of CD68-positivestaining on the various sections was also quantified using MetaMorphsoftware (FIG. 18). The degree of staining in these sections mirroredthe amounts of liver GL1 levels determined biochemically (FIG. 15)further supporting the suggestions on the relative merits of thedifferent treatment regimens.

Example 8: Efficacy of Formula (I) Hemitartrate in a Mouse Model ofGaucher Disease

Animal studies. Procedures involving animals were reviewed and approvedby an Institutional animal care and use committee (IACUC) followingAssociation for assessment and accreditation of laboratory animal care(AAALAC),

State and Federal guidelines. The Gaucher gba^(D409V/null) mice (SeeY.-H. Xu. et al., Am. J. Pathol. 163 (2003) 2093-2101, the entireteachings of which are incorporated herein by reference) were allowed tomature according to study requirements. No difference in phenotype orresponse to Formula (I) Hemitartrate has been found between males andfemales, so both sexes were used in the studies. Formula (I)Hemitartrate delivery was by a single daily oral gavage at a volume of10 mL/kg. Animals were acclimated to oral gavaging with a similar volumeof water for one week prior to initiation of treatment. Formula (I)Hemitartrate was dissolved in Water For Injection (WFI; VWR, WestChester, Pa.) and administered in a dose escalation from 75 mg/kg/day to150 mg/kg/day over the course of nine days, with three days at each doseand increments of 25 mg/kg/day. Mice were weighed three times per weekto monitor the potential impact of the drug on their overall health.Animals were killed by carbon dioxide inhalation and their tissuesharvested immediately. Half of each tissue was snapped frozen on dry iceand stored at −80° C. until ready for further processing. The other halfwas collected for histological analysis.

Quantitation of tissue glucosylceramide levels by high performance thinlayer chromatography. High performance thin layer chromatography(HP-TLC) analysis were as described (A. Abe, et al., J. Clin. Inv. 105(2000) 1563-1571; H. Zhao, et al. Diabetes 56 (2007) 1341-1349; and S.P. F. Miller, et al. J. Lab. Clin. Med. 127 (1996) 353-358, the entireteachings of each are incorporated herein by reference). Briefly, atotal lipid fraction was obtained by homogenizing tissue in cold PBS,extracting with 2:1 (v/v) chloroform:methanol. and sonicating in a waterbath sonicator. Samples were centrifuged to separate the phases and thesupernatant was recovered. The pellets were re-sonicated inchloroform:methanol:saline, centrifuged and the resulting secondsupernatant was collected and combined with the first. A 1:1 (v/v)chloroform:saline mixture was aided to the combined supernatants,vortexed, and centrifuged. After discarding the upper aqueous layer,methanol:saline was added, vortexed and re-centrifuged. The organicphase was taken and dried under nitrogen, dissolved in 2:1 (v/v)chloroform:methanol at 1 mL per 0.1 g original tissue weight and storedat −20° C.

A portion of the lipid extract was used to measure total phosphate, (SeeB. N. Ames, Methods Enzymol. 8 (1966) 115-118, the entire teachings ofwhich are incorporated herein by reference), i.e., the phospholipidcontent to use as an internal standard. The remainder underwent alkalinemethanolysis to remove phospholipids that migrate with glucosylceramideon the HP-TLC plate. Aliquots of the extracts containing equivalentamounts of the total phosphate were spotted onto a HP-TLC plate alongwith known glucosylceramide standards (Matreya inc. Pleasant Gap, Pa.).The lipids were resolved and visualized with 3% cupric acetatemonohydrate (w/v). 15% phosphoric acid (v/v) followed by baking for 10min at 150° C. The lipid bands were scanned on a densitometer (GS-700,Bio-Rad, Hercules, Calif.) and analyzed by Quantity One software(Bio-Rad).

Quantitation of tissue glucosylceramide levels by mass spectrometry.Glucosylceramide was quantified by mass spectrometry as described. (SeeK. McEachern, et al. J. Gene Med. 8 (2006) 719-729; T. Doering, et al.,J. Biol. Chem. 274 (1999) 11038-11045, the entire teachings of each areincorporated herein by reference). Tissue was homogenized in 2:1 (v/v)chloroform:methanol and incubated at 37° C. Samples were centrifuged andthe supernatants were extracted with 0.2 volumes of water overnight. Thesamples were centrifuged again, the aqueous phase was discarded, and theorganic phase dried down to a film under nitrogen.

For electrospray ionization mass spectrometry (ESI/MS) analysis, tissuesamples were reconstituted to the equivalent of. 50 ng original tissueweight in 1 mL chloroform/methanol (2:1, v/v) and vortexed for 5 min.Aliquots of each sample (40 μL) were delivered to Waters total recoveryvials and 50 μL a 10 μg/mL d3-C16-GL-1 internal standard (Matreya, Inc.,Pleasant Gap, Pa.) was added. Samples were dried under nitrogen andreconstituted with 200 μL of 1:4 DMSO:methanol. ESI/MS analysis ofglucosylceramides of different carbon chain lengths was performed on aWaters alliance HPLC (Separation Module 2695) coupled to a MicromassQuattro Micro system equipped with an electrospray ion source. Twentymicroliter lipid extract samples were injected on a C8 column (4 ml×3 mmi.d; Phenomenex, Torrance, Calif.) at 45° C. and eluted with a gradientof 50-100% acetonitrile (2 mM ammonium acetate, 0.1% formic acid) at 0.5mL/min. The first 0.5 min are held at 50% organic and then quicklyswitched to 100% for the final 3.5 min. The source temperature was heldconstant at 150° C. and nitrogen was used as the desolvation gas at aflow rate of 670 L/h. The capillary voltage was maintained at 3.80 KVwith a cone voltage of 23 V, while the dwell time for each ion specieswas 100 ms. Spectra were acquired by the MRM mode to monitor eightdominant isoforms (C16:0, C18:0, C20:0, C22:1. C22:0. C22:1-OH, C24:1,and C24:0). Quantitation of glucosylceramide is based on the sum ofthese eight isoforms to the internal standard, with a calibration curverange from 0.1 to 10 μg/mL.

Histology. For histological analysis, tissues were fixed in zincformalin (Electron Microscopy Sciences, Hatfield, Pa.) at roomtemperature for 24 h, then stored in PBS at 4° C. until ready forfurther processing. All samples were dehydrated in ascendingconcentrations of alcohol, cleared in xylenes and infiltrated andembedded in Surgipath R paraffin (Surgipath, Richmond, Ill.). Fivemicron sections were cut using a rotary microtome and dried in a 60° C.oven prior to staining. Sections were deparaffinized in xylenes, andrehydrated in descending concentrations of alcohol followed by a waterwash. After a 1 min rinse in 3% acetic acid, slides were stained for 40min in 1% Alcian Blue 8GX (Electron Microscopy Sciences) in 3% aceticacid pH 2.0. After rinsing in water and oxidizing in 1% periodic acidfor 1 min. slides were stained with Schiff's reagent (Surgipath) for 12min. After washing for 5 min in hot water, the slides were dehydrated inalcohol and cleared in xylenes prior to mounting with SHUR/Mount™coverglass mounting medium (TBS, Durham, N.C.). Gaucher cells identifiedmorphologically in the liver were quantified using a manual cell countper 10 high power fields (HPFs, 400×).

Results

Effect of administering of Formula (I) Hemitartrate to D409V null mice.The effect of administering Formula (I) Hemitartrate to D409V/null micewas assessed. Approximately 7-month-old mice were administered 150mg/kg/day Formula (I) Hemitartrate (a dose shown in preliminary studiesto be effective at inhibiting glucosylceramide synthase) by oral gavagefor 10 weeks. This treatment had no notable effects on the well-being orfeeding habits of the mice. Measurements of their body weight throughoutthe study showed no significant deviation from those of untreated micesuggesting that Formula (I) Hemitartrate was well tolerated at a doseshown to be effective at inhibiting the synthase.

Efficacy of Formula (I) Hemitartrate at treating young, pre-symptomaticGaucher, mice. Formula (I) Hemitartrate was evaluated for abatement ofthe lysosomal accumulation of glucosylceramide and the appearance ofGaucher cells in young (10-week old) D409V/null mouse. These youngGaucher mice exhibit low levels of GL-1 in the affected tissues.Ten-week-old animals were administered either 75 or 150 mg/kg/day ofFormula (I) Hemitartrate by oral gavage for 10 weeks. Measurement ofglucosylceramide levels showed a dose-dependent reduction when comparedto age-matched vehicle-treated controls. In the cohort that had beentreated with 150 mg/kg/day, glucosylceramide levels were 60, 40 and 75%of those in the controls, in the liver, lung and spleen, respectively(FIG. 6). The statistically significantly lower levels ofglucosylceramide observed in the liver and lung of treated D409V/nullmice indicated that Formula (I) Hemitartrate was effective at reducingthe accumulation of this glycosphingolipid in these tissues.

Histopathological evaluation of the livers of untreated D409V/null miceat the end of the study (20 weeks of age) showed the presence of Gauchercells throughout the liver. Mice treated with 150 mg/kg/day of Formula(I) Hemitartrate for 10 weeks showed only the occasional presence ofGaucher cells that were also invariably smaller in size. Quantitation ofthese cells in a number of different sections confirmed that thefrequency of Gaucher cells were significantly lower in the Formula (I)Hemitartrate-treated mice. Together, these biochemical and histologicalfindings suggested that daily oral administration of Formula (I)Hemitartrate to pre-symptomatic Gaucher mice was effective at decreasingthe accumulation of glucosylceramide in the affected tissues and theconsequent formation of Gaucher cells in the liver.

Efficacy of Formula (I) Hemitartrate in treating older Gaucher mice withpre-existing pathology. The efficacy of Formula (I) Hemitartrate atarresting or reversing disease progression in older, symptomatic Gauchermice was also evaluated. Seven-month old D409V/null mice wereadministered 150 mg/kg/day Formula (I) Hemitartrate by oral gavage for10 weeks. Analyses of glucosylceramide levels in the liver, lung andspleen of treated mice at 5 and 10 weeks post-treatment showed they hadnot increased beyond those observed at the start of the study. Alter 10weeks of treatment, glucosylceramide levels were determined to be 60%lower in liver, 50% lower in lung and 40% lower in spleen than invehicle-treated mice. These results showed that Formula (I) Hemitartratewas effective at inhibiting the further accumulation of glucosylceramidein mice with an existing burden of storage pathology.

Histopathological analysis of tissue sections showed a reduced number ofGaucher cells in the liver of treated D409V/null mice when compared tountreated controls. Quantitation of the number of Gaucher cellscorroborated the biochemical findings; treated D409V/null mice displayedGaucher cell counts that were not significantly different from those atthe beginning of treatment at both the 5- and 10-week time points.Gaucher cell numbers at both these time points were significantly lowerthan those of untreated D409V/null mice. Together, these datademonstrate that Formula (I) Hemitartrate effectively inhibited furtherglucosylceramide accumulation and Gaucher cell development in animalswith pre-existing pathology.

Discussion

Formula (I) Hemitartrate demonstrated a high degree of specificity forthe enzyme glucosylceramide synthase. There was also no measurableinhibition of glucocerebrosidase activity at the effective dose, whichis an important feature when treating Gaucher disease type 1 patients,the majority of whom retain residual glucocerebrosidase activity. At theeffective dose of 150 mg/kg/day, there were no observablegastro-intestinal issues and there was no difference in body weightsbetween the treated and control untreated groups. Serum concentrationsat and above the IC₅₀ (24-40 nM) were readily attainable with oral dosesthat were below the maximum tolerated level. Formula (I) Hemitartratealso was readily metabolized and cleared: both parent compound andmetabolites effectively cleared within 24 h as shown in single andrepeat oral dose ADME studies with 14C-radiolabelled compound in ratsand dogs.

Using a non-optimized dosing regimen of a single daily oral gavagesuccessfully prevented glucosylceramide accumulation in both young,pre-symptomatic mice and in older Gaucher mice that already exhibitedstorage pathology. The young, 10-week old mice, although harboringelevated glucosylceramide levels relative to wild-type controls, had notyet developed the characteristic engorged tissue macrophages, termedGaucher cells. Treatment with 150 mg/kg/day of Formula (I) Hemitartratehalted all measurable disease progression and inhibited the developmentof Gaucher cells. In older mice exhibiting a higher level of lysosomalglucosylceramide and number of Gaucher cells, there was no furtherincrease in the levels of the glycosphingolipid or in the number ofstorage cells after either 5 weeks or 10 weeks of treatment. As themajor source of glucosylceramide in Gaucher cells is reported to beextracellular in origin these results implied that Formula (I)Hemitartrate inhibition of glucosylceramide synthase was systemic.

The observation that Formula (I) Hemitartrate was effective inpreventing further accumulation of glucosylceramide suggests atherapeutic strategy that could further enhance the treatment of Gaucherdisease.

In summary, the data presented here demonstrated that Formula (I)Hemitartrate is an active and specific inhibitor of glucosylceramidesynthase exhibiting no overt adverse effects in a mouse model of Gaucherdisease. It successfully prevented disease progression in bothpre-symptomatic and older diseased Gaucher mice by inhibitingglucosylceramide accumulation and Gaucher cell formation. These findingssuggest that Formula (I) Hemitartrate may represent yet anothertherapeutic option for both pediatric and adult Gaucher type 1 diseaseand potentially other glycosphingolipid storage disorders.

Example 9: Phase 2 Clinical Trial of Formula (I) Hemitartrate

Method. This clinical trial of Formula (I) Hemitartrate, given 50 or 100mg bid orally, treated 26 adults with Gaucher disease type 1 (GD1)(16F:10M; mean age of 34 years, range 18-60; all Caucasian) at 7 sitesin 5 countries. Patients were to have splenomegaly (volume 10 normal)and either thrombocytopenia (platelets 45,000-100,000/mm³) or anemia(hemoglobin 8-10 g/dl, female; 8-11 g/dl, male). None received enzymereplacement or substrate reduction therapy in the prior 12 months. Thecomposite primary efficacy endpoint is globin level (+0.5 g/dl) orplatelet count (+15%) after 52 weeks of treatment. Liver volume,chitotriosidase, glucosylceramide are also assessed. Patients continueto be treated and monitored long-term.

Results. Week 52 data were available for up to 20 patients: 4 otherswithdrew prematurely and 2 were ongoing. The composite primary endpointwas met by 19 of the 20 patients. Mean (1SD) changes from baseline toWeek 52 were: hemoglobin+1.6 (11.35) g/dL; platelet count+43.6%(137.59%); spleen and liver volume (multiples of normal) 40.2% (110.44%)and 15.8% (110.39%), respectively; and chitotriosidase 49.9% (120.75%).Plasma glucosylceramide levels normalized after 4 weeks in all patients,Formula (I) Hemitartrate was well tolerated with an acceptable safetyprofile. Seven related adverse events in 6 patients have been reportedas related; all were mild and transient in nature.

Example 10: Formula (I) Hemitartrate Pharmaceutical Composition, 100 mgCapsules

Method of Preparation of 100 ng Capsules: Formula (I) Hemitartrate,microcrystalline cellulose, lactose monohydrate, and hypromellose, E15were separately passed through a 20 mesh screen. Amounts of the screenedingredients indicated in Table 9 were blended in a high-shear granulatorfor nine to twelve minutes.

TABLE 9 Pharmaceutical Formulation for 100 mg Capsules Unit AmountNominal Batch Unit Amount % per Unit Size: 71,000 100 mg Dose CapsulesTotal Ingredient Capsule (mg) % w/w) Quantity 19.2 kg Formula (I)Hemitartrate 100.0 37.0 7.1 Microcrystallines cellulose 45.0 16.7 3.2Lactose monohydrate 111.5 41.3 7.9 Hypromellose, E15 10.8 4.0 0.8Glyceryl behenate 2.7 1.0 0.2 Filled weight (mg) 270 248-292 mg Total %composition 100.0 19.2 kg

The ingredients were then wet granulated by the addition of purifiedwater (2.2 kg; 11.7% of dry ingredients' weight) to the granulator bowluntil completion, as visually confirmed. The wet granulation wasdischarged from the bowl and passed through a rotating impellor,screening mill. The wet granulation was then dried in a direct heating,static, solid, bed, tray dry oven at 50±5° C. to moisture content of notmore than 3.5%, as confirmed by in-process check. The dry granules werethen passed through a screening mill and the screened granules weretransferred to a V-blender. Glyceryl behenate (0.2 kg) was added to theV-blender, and the final blend was mixed until the blend was uniform, asdetermined by an in-line or off-line blend uniformity test, typicallyfor ten to twenty minutes. The final blend was then encapsulated in a#2-size capsule using a semi-automatic capsule filler to the appropriatefill weight (270 mg average), and the filled capsules were dedustedbefore packaging.

Example 11A: Formula (I) Hemitartrate Pharmaceutical Composition, 10 mgCapsules

Method of Preparation of 10 mg Capsules: The procedure of Example 10 wasfollowed up to the encapsulation step. To produce a 10 mg capsule, thefinal blend was encapsulated in a #4 or #5-size capsule using a capsulefilling machine to the appropriate fill weight (27 mg average), and thefilled capsules were dedusted before packaging.

Example 11B: Formula (I) Hemitartrate Pharmaceutical Composition, 50 mgCapsules

Method of Preparation of 50 mg Capsules: The procedure of Example 10 wasfollowed up to the encapsulation step. To produce a 50 mg capsule, thefinal blend was encapsulated in a #3-size capsule using a capsulefilling machine to the appropriate fill weight (135 mg average), and thefilled capsules were dedusted before packaging.

Example 11C: Formula (I) Hemitartrate Pharmaceutical Composition, 150 mgCapsules

Method of Preparation of 150 mg Capsules: The procedure of Example 10was followed up to the encapsulation step. To produce a 150 mg capsule,the final blend was encapsulated in a #0-size capsule using a capsulefilling machine to the appropriate fill weight (405 mg average), and thefilled capsules were dedusted before packaging.

Example 12: Formula (I) Hemitartrate Pharmaceutical Composition, 25 mgCapsules

Method of Preparation of 25 mg Capsules: The procedure of Example 10 wasfollowed up to the encapsulation step. To produce a 25 mg capsule, thefinal blend was encapsulated in a #4-size capsule using a capsulefilling machine to the appropriate fill weight (67.5 mg average), andthe filled capsules were dedusted before packaging.

Example 13: Formula (I) Hemitartrate Drug Interactions—CYP2D6 Inhibitors

A study was performed to evaluate the pharmacokinetics, safety andtolerability of multiple oral doses of Formula (I) Hemitartrate (100 mgBID) administered with and without paroxetine (30 mg once daily), apotent inhibitor of CYP2D6. This was an open-label, fixed-sequence studyin 36 healthy subjects (17 males and 19 females). The secondaryobjectives were to evaluate the PK of paroxetine in combination withmultiple doses of Formula (I) Hemitartrate (100 mg BID) in healthysubjects and to further evaluate Formula (I) Hemitartrate PK followingmultiple-dose compared with single-dose Formula (I) Hemitartrateadministration.

The mean PK parameters of the free base of Formula (I) Hemitartrate asit exists in plasma were nonlinear and showed a 2-fold accumulation inAUC and C_(max) with repeated administration (100 mg BID) as compared tosingle dose administration. Concomitant administration of Formula (I)Hemitartrate and paroxetine resulted in a 7-fold increase in C_(max) and9-fold increase in AUC as compared to the multiple-dose administrationof Formula (I) Hemitartrate alone. These results indicate thatparoxetine can inhibit the metabolism of Formula (I) Hemitartrate andincreases blood plasma concentrations of the drug. Similar effects wouldbe expected with other potent CYP2D6 inhibitors (e.g. fluoxetine andquinidine) and careful monitoring of drug plasma levels and potentialdose adjustments are necessary when Formula (I) Hemitartrate isco-administered with a drug known to be a potent CYP2D6 inhibitor.Paroxetine concentrations were about 1.5- to 2-fold higher than expectedwhich suggests that Formula (I) Hemitartrate or one of its metabolitesmay be a mild inhibitor of CYP2D6.

Example 14: Formula (I) Hemitartrate Drug Interactions—CYP3A4 Inhibitorsand p-Glycoprotein (PGP) Inhibitors

A study was performed to evaluate the pharmacokinetics, safety, andtolerability of multiple doses of Formula (I) Hemitartrate (100 mg twicedaily) with and without multiple-dose ketoconazole (400 mg once daily)in healthy male and female subjects. This was an open-labelfixed-sequence study in 36 healthy subjects (18 males and females)consisting of 3 periods which included 100-mg single-dose administrationof Formula (I) Hemitartrate, multiple-dose administration of Formula (I)Hemitartrate, and concomitant administration of Formula (I) Hemitartrate100 mg (twice daily) with ketoconazole 400 mg (once daily). Repeatedadministration of Formula (I) Hemitartrate and ketoconazole, a stronginhibitor of Cytochrome p450 3A4 (“CYP 3A4”) and p-glycoprotein,resulted in a 4-fold increase in exposure of the free base of theFormula (I) Hemitartrate as it exists in plasma at steady state. Thus,patients already receiving Formula (I) Hemitartrate may require atemporary dose reduction while on concomitant therapy with stronginhibitors of CYP 3A4 or p-glycoprotein.

Example 15—Stability Studies for Formula (I) Hemitartrate Formulation

Blends were prepared by mixing Formula (I) Hemitartrate and excipients(Lactose Monohydrate capsulating grade, Avicel PH 301 (Microcrystallinecellulose) and Methocel E15 Prem LV (Hydroxypropylmethylcellulose) in ascintillation vial at about a two gram scale. 15.6% water was added tothe blend and mixed to form wet granules. The wet granules were screenedusing a #10 sieve (opening of 2000 microns). The screened granules werethen dried in an oven at 50° C. for 2 hours. The dried granules werescreened using a #18 sieve (opening of 1000 microns). The lubricant,glyceryl behenate, was added to the blend and mixed to form the finalblend. The blends prepared are shown in the table below:

TABLE 10 Avicel Lactose PH 50 mg/100 mg Lot # AP Monohydrate 101Formulation Comment 1 1 2.1 2.1 50 control 2 1 2.1 0 50 without Avicel 31 0 2.1 50 without Lactose 4 1 2.1 1.1 50 less Avicel 5 1 1.1 2.1 50less Lactose 6 1 2.1 0.8 50 Avicel and Lactose ratio comparable to 100mg 7 1 1.1 0.4 100 controlMethocel (HPMC) was used in the range of 2 to 4%Compritol ATO 88 was used in the range of 1 to 1.6%

The seven formulation blends, which have different API:lactose:Avicelratios, listed above were exposed to a high temperature at 85° C. for 3days (a forced-degradation study condition) in order to understand thedegradation rate and the stability of each formulation. This acceleratedcondition was chosen based on the study results that the extent of thedegradation products of the 50 mg drug product at 24 months was similarto those obtained at 85° C. for 3 days.

The forced-degradation study was performed using a reverse phasegradient HPLC method which used a C18 column (Waters T3, 3 μm, 100×4.6mm), mobile phases consisting of water and acetonitrile with 0.1%trifluoroacetic acid (TFA), UV detection at 280 nm, column temperatureat 40° C., and flow rate at 2 mL/min. The gradient started at holding at5% B (acetonitrile and 0.1% TFA) for 0.5 minutes, and then ramping uporganic component at 4.83% B per minute up to 15 minutes.

The total degradants of each formulation blend was summed and plottedagainst the ratio of API:Lactose:Avicel and the results are shown inFIG. 15. The study results suggest that while keeping the API andLactose ratio constant, decreasing the amount of avicel improves thestability of the formulation. When avicel is removed, the formulationhas API:Lactose:Avicel ratio of 1:2.1:0, it is the most stableformulation. When the lactose is removed, the formulation has aAPI:Lactose:Avicel Ratio of 1:0:2.1, and this formulation is not themost unstable comparing to other ratios. The combined informationsuggests that lactose stabilizes the formulation, while aviceldestabilizes the formulation. However, when both excipients are present,they interact with each other. The ratio most be adjusted to obtain astable formulation.

For active pharmaceutical ingredients like Formula (I) hemihydrate thatare water soluble, microcrystalline cellulose helps to form granulesduring wet granulation as it is insoluble in water. If microcrystallinecellulose was not used, a sharp change occurs from the granule stage toa paste form. The paste form was difficult to handle and the resultingparticles after drying do not have the suitable mechanical strength andparticle size distribution. The pharmaceutical composition that has 37wt % of a Formula (I) Hemitartrate, 41.0 wt % of a water-soluble filler;16.7 wt % of an insoluble filler, 2 wt % to about 6 wt % of a binder;and about 0.1 wt % to about 2 wt % of a lubricant, all on a dry solidsbasis has the best stability profile with respect to the amount ofdegradants formed.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this has been particularly shown and described with references toexample embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the encompassed by the appendedclaims.

1-93. (canceled) 94: A method of treating a human patient with type 1Gaucher disease who has been assessed as being a poor CYP2D6 P450metabolizer as determined by CYP2D6 genotyping comprising administeringto said patient an effective amount of a hemitartrate salt of thecompound of formula (I)

wherein the effective amount is 100 milligrams. 95: The method of claim94, wherein the effective amount of the hemitartrate salt of thecompound of formula (I) is administered in an oral dosage form once perday. 96: The method of claim 95, wherein the hemitartrate salt of thecompound of formula (I) is in crystalline Form A. 97: The method ofclaim 96, wherein the crystalline Form A is characterized by at leastthree X-ray powder diffraction peaks at 2θ angles selected from thegroup consisting of 5.1°, 6.6°, 10.7°, 11.0°, 15.9°, and 21.7°, whereinthe specified 2θ angle means the specified value±0.2°. 98: The method ofclaim 97, wherein the X-ray powder diffraction peaks are obtained byusing Cu Kα radiation. 99: The method of claim 95, wherein the oraldosage form is a capsule. 100: The method of claim 95, wherein thehemitartrate salt of the compound of formula (I) is in crystalline formhaving at least three X-ray powder diffraction peaks at 20 anglesselected from the group consisting of 5.1°, 6.6°, 10.7°, 11.0°, 15.9°,and 21.7°, wherein the specified 2θ angle means the specifiedvalue±0.2°. 101: The method of claim 100, wherein the X-ray powderdiffraction peaks are obtained by using Cu Kα radiation. 102: A methodof treating a human patient with type 1 Gaucher disease who has beenassessed as being an intermediate CYP2D6 P450 metabolizer as determinedby CYP2D6 genotyping comprising administering to said patient aneffective amount of a hemitartrate salt of the compound of formula (I)

wherein the effective amount is 100 milligrams administered in an oraldosage form twice per day. 103: The method of claim 102, wherein thehemitartrate salt of the compound of formula (I) is in crystalline FormA. 104: The method of claim 103, wherein the crystalline Form A ischaracterized by at least three X-ray powder diffraction peaks at 2θangles selected from the group consisting of 5.1°, 6.6°, 10.7°, 11.0°,15.9°, and 21.7°, wherein the specified 2θ angle means the specifiedvalue±0.2°. 105: The method of claim 104, wherein the X-ray powderdiffraction peaks are obtained by using Cu Kα radiation. 106: The methodof claim 102, wherein the oral dosage form is a capsule. 107: The methodof claim 102, wherein the hemitartrate salt of the compound of formula(I) is in crystalline form having at least three X-ray powderdiffraction peaks at 2θ angles selected from the group consisting of5.1°, 6.6°, 10.7°, 11.0°, 15.9°, and 21.7°, wherein the specified 2θangle means the specified value±0.2°. 108: A method of treating a humanpatient with type 1 Gaucher disease who has been assessed as being anextensive CYP2D6 P450 metabolizer as determined by CYP2D6 genotypingcomprising administering to said patient an effective amount of ahemitartrate salt of the compound of formula (I)

wherein the effective amount is 100 milligrams administered in an oraldosage form twice per day. 109: The method of claim 108, wherein thehemitartrate salt of the compound of formula (I) is in crystalline FormA. 110: The method of claim 109, wherein the crystalline Form A ischaracterized by at least three X-ray powder diffraction peaks at 2θangles selected from the group consisting of 5.1°, 6.6°, 10.7°, 11.0°,15.9°, and 21.7°, wherein the specified 2θ angle means the specifiedvalue±0.2°. 111: The method of claim 110, wherein the X-ray powderdiffraction peaks are obtained by using Cu Kα radiation. 112: The methodof claim 108, wherein the oral dosage form is a capsule. 113: The methodof claim 108, wherein the hemitartrate salt of the compound of formula(I) is in crystalline form having at least three X-ray powderdiffraction peaks at 20 angles selected from the group consisting of5.1°, 6.6°, 10.7°, 11.0°, 15.9°, and 21.7°, wherein the specified 2θangle means the specified value±0.2°.